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(57) Abstract: In order to identify an antigen to 2D7 antibody, a 2D7 antigen is cloned. As a result, it. is indicated that the 2D7
ON antigen is an HLA class I molecule. Based on this finding, it is discussed whether the 2D7 antibody has an activity of inducing cell
OS death or not. As a result, nuclear fragmentation is observed by further crosslinking the 2D7 antibody with another antibody, thereby
indicating that cell death is induced. It is also clarified that a diabody of the 2D7 antibody has an extremely strong cell death-inducing
activity even though no other antibody is added. These results point out that a degradation product of an antibody recognizing HLA
is usable as a cell death-inducing agent.
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10 v\
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[^#fF^3] Genestier et al. , Blood 90: 726-735 (1997)
C^WiE&iU] Genestier et al. , J. Biol. Chem. 273: 5060-5066 (1998)
20 ffitfc&XWtS') Woodle et al. , J. Immunol. 158: 2156-2164 (1997)
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10° 10 1 10 2
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WO 2004/033499
PCT/JP2003/013063
WO 2004/033499
PCT/JP2003/013063
1 5 A
2 2/3 7
1 5 B
PI Log
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WO 2004/033499
PCT/JP2003/013063
1 5C
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WO 2004/033499
PCT/JP2003/013063
1 6 A
10°
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10 2 -J
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12.6%
2 4/3 7
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10 2
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2.0 ug/ml
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1 6 B
10°
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11.9%
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FITC Log
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WO 2004/033499
PCT/JP2003/013063
mi 7 a
2 5/3 7
10°
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WO 2004/033499
PCT/JP2003/013063
Ml 7B
2 6/3 7
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FITC Log
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10
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FITC Log
WO 2004/033499
PCT/JP2003/013063
1 8 A
2 7/3 7
1 8 B
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PI Log
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WO 2004/033499
PCT/JP2003/013063
2 8/3 7
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WO 2004/033499
PCT/JP2003/013063
0 2 0
2 9/3 7
PI Log
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WO 2004/033499
PCT/JP2003/013063
3 0/3 7
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Q Q
WO 2004/033499
PCT/JP2003/013063
3 1/3 7
02 2
0 200 500 1000
2D7DB ( ng/ml)
WO 2004/033499
PCT/JP2003/013063
3 2/3 7
M2S
2D7DB
15#
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WO 2004/033499
PCT/JP2003/013063
3 3/3 7
WO 2004/033499
PCT/JP2003/013063
3 4/3 7
m2 5
0 1 1 1 1 1 — ■ ' " 1
0 20 40 60 80 100 120 140
Vehicle 2D7 diabody
WO 2004/033499
PCT/JP2003/013063
3 5/3 7
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Z0021195.LMD
10 u
10' 10'
FITC
10° 10"
UL
UR
LL
LR
4.94
20.26
65.33
9.47
O.
CM
o,
o
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10 u
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File:
Z0021198.LMD
Quad % Total
10' 10 2 10 3
FITC
■KIWI
10
UL
UR
LL
LR
2.04
22.04
65.98
9.94
10 10'
FITC
7^
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File:
Z0021194.LMD
Quad % Total
7.23
45.44
36.01
11.32
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CO
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CM
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o
o.
■
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10 u
10' 10 2 10 3
FITC
10'
File:
Z0021197.LMD
Quad % Total
UL
UR
LL
LR
2.37
30.29
52.65
14.69
10 u
10' 10'
FITC
10
Mr]
10"
File:
Z0021193.LMD
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42.35
40.76
10.00
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File:
Z0021196.LMD
Quad % Total
UL
UR
LL
LR
3.16
33.60
48.20
15.04
FITC
WO 2004/033499
PCT/JP2003/013063
3 6/3 7
2 6C
2 6D
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10 u
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10' 10'
FITC
10 10'
File:
Z0021202.LMD
Quad % Total
UL
UR
LL
LR
2.07
31.24
56.25
10.44
File:
Z0021205.LMD
Quad % Total
10° 10 1 10 2 10 3 10 4
FITC
UL
UR
LL
LR
1.20
28.74
63.74
6.32
o.
File:
Z0021201.LMD
1.98
37.69
30.44
29.89
FITC
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FITC
m
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CM
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10° 10 1 10 2 10 3 10'
FITC
File:
Z0021204.LMD
Quad % Total
UL
UR
LL
LR
0.91
22.71
40.28
36.10
File:
2r
Z0021199.LMD
Quad % Total
UL 2.46
cm :
UR 40.37
LL 31 .00
o.
LR 26.17
O ■
10° 10 1 10 2 10 3 10'
FITC
File:
Z0021203.LMD
Quad % Total
UL
UR
LL
LR
0.93
24.60
42.03
32.44
WO 2004/033499
PCT/JP2003/013063
3 7/3 7
M2 6E
File: 20021 208.LMD
Quad
% Total
UL
1.95
UR
15.84
LL
77.05
LR
5.16
10" 10 10" 10 10*
FITC
o
CM
_o
T^"
• - . .
File:Z0021207.LMD
Quad
% Total
UL
1.51
UR
55.82
LL
27.05
LR
15.62
FITC
o
to
O
CM
_o
m
• Quad % Total
2"! lllhq
10" 10
File: Z0021206.LMD
UL
UR
LL
LR
10 2 10 3 10'
1.40
57.54
20.64
20.42
FITC
WO 2004/033499
PCT/JP2003/013063
1/1 7
SEQUENCE LISTING
<110> CHUGAI SEIYAKU KABUSHIKI KAISHA
OZAKI Shuji
ABE Masahiro
<120> Inducer Of Cell Death
<130> C1-A0220P
<140>
<141>
<150> JP 2002-299289
<151> 2002-10-11
<160> 14
<170> Patentln Ver. 2. 1
<210> 1
<211> 547
<212> DNA
<213> Mus musculus
<220>
WO 2004/033499
PCT/JP2003/013063
2/1 7
<221> CDS
<222> (103).. (546)
<400> 1
tacgactcac tatagggcaa gcagtggtat caacgcagag tacgcgggga atctatgatc 60
agtgtcctct ctacacagtc cctgacgaca ctgactccaa cc atg cga tgg age 114
Met Arg Trp Ser
1
tgg ate ttt etc ttc etc ctg tea ata act gca ggt gtc cat tgc cag 162
Trp He Phe Leu Phe Leu Leu Ser He Thr Ala Gly Val His Cys Gin
5 10 15 20
gtc cag ttg cag cag tct gga cct gag ctg gtg aag cct ggg get tea 210
Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser
25 30 35
gtg aag atg tct tgt aag get tct ggc tac acc ttc aca gac tac ttt 258
Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Phe
40 45 50
ata cac tgg gtg aaa cag agg cct gga cag gga'ctt gaa tgg att gga 306
He His Trp Val Lys Gin Arg Pro Gly Gin Gly Leu Glu Trp He Gly
55 60 65
WO 2004/033499
PCT/JP2003/013063
3/17
tgg att ttt cct gga gat gat act act gat tac aat gag aag ttc agg 354
Trp He Phe Pro Gly Asp Asp Thr Thr Asp Tyr Asn Glu Lys Phe Arg
70 75 80
ggc aag acc aca ctg act gca gac aaa tec tec age aca gec tac att 402
Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr lie
85 90 95 100
ttg etc age age ctg acc tct gag gac tct gcg atg tat ttc tgt gta 450
Leu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Met Tyr Phe Cys Val
105 110 115
agg agt gac gac ttt gac tac tgg ggc cag ggc acc act etc aca gtc 498
Arg Ser Asp Asp Phe Asp Tyr Trp Gly Gin Gly Thr Thr Leu Thr Val
120 125 130
tec tea gee aaa aca aca ccc cca tea gtc tat cca ctg gec cct get g 547
Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Ala
135 140 145
<210> 2
<211> 148
<212> PRT
<213> Mus musculus
WO 2004/033499
PCT/JP2003/013063
<400> 2
Met Arg Trp Ser Trp lie Phe Leu
1 5
Val His Cys Gin Val Gin Leu Gin
20
4/17
Phe Leu Leu Ser He Thr Ala Gly
10 15
Gin Ser Gly Pro Glu Leu Val Lys
25 30
Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe
35 40 45
Thr Asp Tyr Phe He His Trp Val Lys Gin Arg Pro Gly Gin Gly Leu
50 55 60
Glu Trp He Gly Trp He Phe Pro Gly Asp Asp Thr Thr Asp Tyr Asn
65 70 75 80
Glu Lys Phe Arg Gly Lys Thr Thr Leu Thr Ala Asp Lys Ser Ser Ser
85 90 95
Thr Ala Tyr lie Leu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Met
100 105 110
Tyr Phe Cys Val Arg Ser Asp Asp Phe Asp Tyr Trp Gly Gin Gly Thr
115 120 125
Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro
WO 2004/033499
PCT/JP2003/013063
5/1 7
130 135 140
Leu Ala Pro Ala
145
<210> 3
<211> 535
<212> DNA
<213> Mus musculus
<220>
<221> CDS
<222> (103) . . (534)
<400> 3
ctaatacgac tcactatagg gcaagcagtg gtatcaacgc agagtacgcg gggactwatg 60
agaatagcag taattagcta gggaccaaaa ttcaaagaca aa atg cat ttt caa 114
Met His Phe Gin
1
gtg cag att ttc age ttc ctg eta ate agt gec tea gtc ate atg tec 162
Val Gin He Phe Ser Phe Leu Leu He Ser Ala Ser Val He Met Ser
5 10 15 20
WO 2004/033499
PCT/JP2003/013063
6/1 7
aga gga caa att gtt etc acc cag teg cca gca ate atg tct gca tct 210
Arg Gly Gin He Val Leu Thr Gin Ser Pro Ala He Met Ser Ala Ser
25 30 35
cca ggg gag aag gtc acc ata acc tgc agt gee age tea agt gta agt 258
Pro Gly Glu Lys Val Thr lie Thr Cys Ser Ala Ser Ser Ser Val Ser
40 45 50
tac atg cac tgg ttc cag cag aag cca ggc act ttt ccc aaa etc tgg 306
Tyr Met His Trp Phe Gin Gin Lys Pro Gly Thr Phe Pro Lys Leu Trp
55 60 65
att tat age aca tec aac ctg get tct gga gtc cct act cgc ttc agt 354
lie Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Thr Arg Phe Ser
70 75 80
ggc agt gga tct ggg acc tct tac tct etc aca ate age cga atg gag 402
Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr He Ser Arg Met Glu
85 90 95 100
get gaa gat get gee act tat tac tgc cag caa agg acg agt tat cca 450
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Arg Thr Ser Tyr Pro
105 110 115
ccc acg ttc ggc teg ggg aca aag ttg gag ata aaa egg get gat get 498
Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu He Lys Arg Ala Asp Ala
WO 2004/033499
PCT/JP2003/013063
7/17
120 125 130
gca cca act gta tec ate ttc cca cca tec agt gag c 535
Ala Pro Thr Val Ser He Phe Pro Pro Ser Ser Glu
135 140
<210> 4
<211> 144
<212> PRT
<213> Mus musculus
<400> 4
Met His Phe Gin Val Gin He Phe Ser Phe Leu Leu He Ser Ala Ser
15 10 15
Val He Met Ser Arg Gly Gin He Val Leu Thr Gin Ser Pro Ala He
20 25 30
Met Ser Ala Ser Pro Gly Glu Lys Val Thr He Thr Cys Ser Ala Ser
35 40 45
Ser Ser Val Ser Tyr Met His Trp Phe Gin Gin Lys Pro Gly Thr Phe
50 55 60
Pro Lys Leu Trp He Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro
WO 2004/033499
PCT/JP2003/013063
8/1 7
65 . 70 75 80
Thr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr lie
85 90 95
Ser Arg Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Arg
100 105 110
Thr Ser Tyr Pro Pro Thr Phe Gly Ser Gly Thr Lys Leu Glu He Lys
115 120 125
Arg Ala Asp Ala Ala Pro Thr Val Ser lie Phe Pro Pro Ser Ser Glu
130 135 140
<210> 5
<211> 789
<212> DNA
<213> Artificial Sequence
<220>
<221> CDS
<222> (14) . . (775)
<223> Description of Artificial Sequence: an artificially
synthesized DNA sequence
WO 2004/033499
PCT/JP2003/013063
9/17
<400> 5
cctgaattcc acc atg cga tgg age tgg ate ttt etc ttc etc ctg tea 49
Met Arg Trp Ser Trp lie Phe Leu Phe Leu Leu Ser
1 5 10
ata act gca ggt gtc cat tgc cag gtc cag ttg cag cag tct gga cct 97
He Thr Ala Gly Val His Cys Gin Val Gin Leu Gin Gin Ser Gly Pro
15 20 25
gag ctg gtg aag cct ggg get tea gtg aag atg tct tgt aag get tct 145
Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser
30 35 40
ggc tac acc ttc aca gac tac ttt ata cac tgg gtg aaa cag agg cct 193
Gly Tyr Thr Phe Thr Asp Tyr Phe He His Trp Val Lys Gin Arg Pro
45 50 55 60
gga cag gga ctt gaa tgg att gga tgg att ttt cct gga gat gat act 241
Gly Gin Gly Leu Glu Trp lie Gly Trp He Phe Pro Gly Asp Asp Thr
65 70 75
act gat tac aat gag aag ttc agg ggc aag acc aca ctg act gca gac 289
Thr Asp Tyr Asn Glu Lys Phe Arg Gly Lys Thr Thr Leu Thr Ala Asp
80 85 90
aaa tec tec age aca gec tac att ttg etc age age ctg acc tct gag 337
WO 2004/033499
PCT/JP2003/013063
10/17
Lys Ser Ser Ser Thr Ala Tyr He Leu Leu Ser Ser Leu Thr Ser Glu
95 100 105
gac tct gcg atg tat ttc tgt gta agg agt gac gac ttt gac tac tgg 385
Asp Ser Ala Met Tyr Phe Cys Val Arg Ser Asp Asp Phe Asp Tyr Trp
110 115 120
ggc cag ggc acc act etc aca gtc tec tea ggt gga ggc ggt age caa 433
Gly Gin Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gin
125 130 135 140
att gtt etc acc cag teg cca gca ate atg tct gca tct cca ggg gag 481
He Val Leu Thr Gin Ser Pro Ala He Met Ser Ala Ser Pro Gly Glu
145 150 155
aag gtc acc ata acc tgc agt gec age tea agt gta agt tac atg cac 529
Lys Val Thr lie Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met His
160 165 170
tgg ttc cag cag aag cca ggc act ttt ccc aaa etc tgg att tat age 577
Trp Phe Gin Gin Lys Pro Gly Thr Phe Pro Lys Leu Trp He Tyr Ser
175 180 185
aca tec aac ctg get tct gga gtc cct act cgc ttc agt ggc agt gga 625
Thr Ser Asn Leu Ala Ser Gly Val Pro Thr Arg Phe Ser Gly Ser Gly
190 195 200
WO 2004/033499
PCT/JP2003/013063
11/17
tct ggg ace tct tac tct etc aca ate age cga atg gag get gaa gat 673
Ser Gly Thr Ser Tyr Ser Leu Thr lie Ser Arg Met Glu Ala Glu Asp
205 210 215 220
get gee act tat tac tgc cag caa agg acg agt tat cca ccc
Ala Ala Thr Tyr Tyr Cys Gin Gin Arg Thr Ser Tyr Pro Pro
225 230
ggg aca aag ttg gag ata aaa gac tac aag gat gac gac gat 769
Gly Thr Lys Leu Glu lie Lys Asp Tyr Lys Asp Asp Asp Asp
240 245 250
aag tga taagcggccg caat 789
Lys
acg ttc 721
Thr Phe
235
ggc teg
Gly Ser
<210> 6
<211> 253
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: an artificially
synthesized peptide sequence
WO 2004/033499
PCT/JP2003/013063
<400> 6
Met Arg Trp Ser Trp
1 5
Val His Cys Gin Val
20
Pro Gly Ala Ser Val
35
Thr Asp Tyr Phe lie
50
Glu Trp He Gly Trp
65
Glu Lys Phe Arg Gly
85
Thr Ala Tyr lie Leu
100
Tyr Phe Cys Val Arg
115
Thr Leu Thr Val Ser
130
Gin Ser Pro Ala He
145
Thr Cys Ser Ala Ser
165
Lys Pro Gly Thr Phe
180
Ala Ser Gly Val Pro
12/17
He Phe Leu Phe Leu Leu
10
Gin Leu Gin Gin Ser Gly
25
Lys Met Ser Cys Lys Ala
40
His Trp Val Lys Gin Arg
55
He Phe Pro Gly Asp Asp
70 75
Lys Thr Thr Leu Thr Ala
90
Leu Ser Ser Leu Thr Ser
105
Ser Asp Asp Phe Asp Tyr
120
Ser Gly Gly Gly Gly Ser
135
Met Ser Ala Ser Pro Gly
150 155
Ser Ser Val Ser Tyr Met
170
Pro Lys Leu Trp He Tyr
185
Thr Arg Phe Ser Gly Ser
Ser He Thr Ala Gly
15
Pro Glu Leu Val Lys
30
Ser Gly Tyr Thr Phe
45
Pro Gly Gin Gly Leu
60
Thr Thr Asp Tyr Asn
80
Asp Lys Ser Ser Ser
95
Glu Asp Ser Ala Met
110
Trp Gly Gin Gly Thr
125
Gin He Val Leu Thr
140
Glu Lys Val Thr He
160
His Trp Phe Gin Gin
175
Ser Thr Ser Asn Leu
190
Gly Ser Gly Thr Ser
WO 2004/033499
PCT/JP2003/013063
13/17
195 200 205
Tyr Ser Leu Thr He Ser Arg Met Glu Ala Glu Asp Ala Ala Thr Tyr
210 215 220
Tyr Cys Gin Gin Arg Thr Ser Tyr Pro Pro Thr Phe Gly Ser Gly Thr
225 230 235 240
Lys Leu Glu He Lys Asp Tyr Lys Asp Asp Asp Asp Lys
245 250
<210> 7
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequenced artificially
synthesized adapter sequence
<400> 7
aattcccagc acagtggtag ataagtaag 29
<210> 8
<211> 29
<212> DNA
<213> Artificial Sequence
WO 2004/033499
PCT/JP2003/013063
14/17
<220>
<223> Description of Artificial Sequence: an artificially
<210> 9
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence:an artificially
synthesized primer sequence
synthesized adapter sequence
<400> 8
tcgacttact tatctaccac tgtgctggg
29
<400>
9
caggggccag tggatagact gatg
24
<210> 10
<211> 23
<212> DNA
<213> Artificial Sequence
WO 2004/033499
PCT/JP2003/013063
15/17
<220>
<223> Description of Artificial Sequence:an artificially-
synthesized primer sequence
<400> 10
<210> 11
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence^an artificially
gctcactgga tggtgggaag atg
23
synthesized primer sequence
<400>
11
cctgaattcc accatgcgat ggagctggat ctttc
35
<210> 12
<211> 47
<212> DNA
<213> Artificial Sequence
WO 2004/033499
PCT/JP2003/013063
16/17
<220>
<223> Description of Artificial Sequence: an artificially-
synthesized primer sequence
<400> 12
aatttggcta ccgcctccac ctgaggagac tgtgagagtg gtgccct 47
<210> 13
<211> 47
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: an artificially
synthesized primer sequence
<400> 13
tcctcaggtg gaggcggtag ccaaattgtt ctcacccagt cgccagc 47
<210> 14
<211> 68
<212> DNA
<213> Artificial Sequence
WO 2004/033499
PCT/JP2003/013063
17/17
<220>
<223> Description of Artificial Sequence: an artificially
synthesized primer sequence
<400> 14
attgcggccg cttatcactt atcgtcgtca tccttgtagt cttttatctc caactttgtc 60
cccgagcc c.o
INTERNATIONAL SEARCH REPORT
International application No.
PCT/JP03/13063
A. CLASSIFICATION OF SUBJECT MATTER
Int. CI 7 C07K16/18, C12P21/08, A61K39/395, A61P35/00, A61P37/02,
A61P43/00
According to International Patent Classification (IPC) or to both national classification and IPC
B. FIELDS SEARCHED
Minimum documentation searched (classification system followed by classification symbols)
Int. CI 7 C07K16/18, C12P21/08, A61K39/395, A61P35/00, A61P37/02,
A61P43/00
Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched
Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)
WPI (DIALOG) , BIOSIS (DIALOG) , JSTPlusu ( JOIS) ,
GeneBank/EMBL/DDBJ/GeneSeq, SwissProt/PIR/GeneSeq
C. DOCUMENTS CONSIDERED TO BE RELEVANT
Category*
Citation of document, with indication, where appropriate, of the relevant passages
Relevant to claim No.
X
A
GENESTIER L. et al., Fas-independent apoptosis of
activated T cells induced by antibodies to the HLA
class I al domain., Blood 1997, Vol.90, No. 9,
pages 3629 to 3639
1
4-
-3
23
X
A
MATSUOKA S . et al . , A novel type of cell death of
lymphocytes induced by monoclonal antibody without
participation of complement., J. Exp .Med. 1995,
Vol.181, No. 6, pages 2007 to 2015
1
4-
-3
•23
X
A
FA YEN J. et al . , Negative signaling by anti-HLA
class I antibodies is dependent upon two triggering
events., Int. Immunol. 1998, Vol.10, No. 9,
pages 1347 to 1358
1
4-
-3
•23
| x 1 Further documents are listed in the continuation of Box C. | | See patent family annex.
* Special categories of cited documents: "T later document published after the international filing date or
"A" document defining the general state of the art which is not priority date and not in conflict with the application but cited to
considered to be of particular relevance undeistand the principle or theory underlying the invention
"E" earlier document but published on or after the international filing "X" document of particular relevance; the claimed invention cannot be
date considered novel or cannot be considered to involve an inventive
"L" document which may throw doubts on priority claim(s) or 'which is step when the document is taken alone
cited to establish the publication date of another citation or other " Y' document of particular relevance; the claimed invention cannot be
special reason (as specified) considered to involve an inventive step when the document is
"O" document referring to an oral disclosure, use, exhibition or other combined with one or more other such documents, such
means combination being obvious to a person skilled in the art
"P" document published prior to the international filing date but later "&" document member of the same patent family
than the priority date claimed
Date of the actual completion of the intemalional search
05 November, 2003 (05.11.03)
Date of mailing of the international search report
18 November, 2003 (18.11.03)
Name and mailing address of the ISA/
Japanese Patent Office
Facsimile No. N
Authorized officer
Telephone No.
Form PCT/ISA/210 (second sheet) (July 1998)
INTERNATIONAL SEARCH REPORT
International application No.
PCT/JP03/13063
C (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT
Category*
Citation of document, with indication, where appropriate, of the relevant passages
Relevant to claim No.
Y
A
Y
A
Y
A
ONO K. et al., The humanized anti-HM1.24 antibody
effectively kills multiple myeloma cells by human
effector cell-mediated cytotoxicity., Mol . Immunol .
1999, Vol.36, No-6, pages 387 to 395
OHTOMO T. et al., Molecular cloning and charac-
terization of a surface antigen preferentially
overexpressed on multiple myeloma cells., Biochem.
Biophys .Res .Commun. 1999, Vol.258, No. 3, pages 583
to 591
OZAKI S. et al., Humanized anti-HM1.24 antibody
mediates myeloma cell cytotoxicity that is enhanced
by cytokine stimulation of effector cells.,
Blood 1999, Vol.93, No. 11, pages 3922 to 3930
1-4
5-23
1-4
5-23
1-4
5-23
Form PCT/ISA/210 (continuation of second sheet) (July 1998)
S^ttlJS#-§- PCT/JP03/1 3 06 3
A. »WOJR-r«»»©«« (BJMMWWB (I PC) )
IntCl' C07K 16/18, C12P 21/08, A61K 39/395, A61P 35/00, A61P 37/02, A61P 43/00
mSttff^jz&wmw mmmfm (ipo > ■
IntCl 7 C07K 16/18, C12P 21/08, A61K 39/395, A61P 35/00, A61P 37/02, A61P 43/00
WPI (DIALOG) , BIOSIS (DIALOG) , JSTPlus (JOIS) , GenBank/EMBL/DDBJ/GeneSeq, SwissProt/PIR/GeneSeq
vmr&
X
GENESTIER L. et al, Fas-independent apoptosis of activated T
1-3
A
cells induced by antibodies to the HLA class I al domain.
4-23
Blood 1997, Vol. 90, No. 9, p. 3629-3639
X
MATSU0KA S. et al,A novel type of cell death of lymphocytes
1-3
A
induced by a monoclonal antibody without participation of
4-23
complement.
J. Exp. Med. 1995, Vol. 181, No. 6, p. 2007-2015
0 CIOitt^5:Wjf$JiTVv5„
05.11.03
B^S^fF/r (ISA/JP)
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.11,03
03-3581-1101
4N
9 15 2
3448
IitPCT/1 SA/2 1 0 (1 9 9 8^7^)
PCT/JP0 3/1 3 06 3
X
A
Y
A
Y
A
Y
A
FAYEN J. et al, Negative signaling by anti-HLA class I
antibodies is dependent upon two triggering events.
Int. Immunol. 1998, Vol. 10, No. 9, p. 1347-1358
ON0 K. et al, The humanized anti-HMl. 24 antibody effectively
kills multiple myeloma cells by human effector cell-mediated
cytotoxicity. Mol. Immunol. 1999, Vol. 36, No. 6, p. 387-395
OHT0MO T. et al, Molecular cloning and characterization of a
surface antigen preferentially overexpressed on multiple
myeloma cells.
Biochem. Biophys. Res. Commun. 1999, Vol. 258, No. 3, p. 583-591
OZAKI S. et al, Humanized anti-HMl. 24 antibody mediates
myeloma cell cytotoxicity that is enhanced by cytokine
stimulation of effector cells.
Blood 1999, Vol. 93, No. 11, p. 3922-3930
1-3
4-23
1-4
5-23
1-4
5-23
1-4
5-23
ttl^PCT/I SA/2 1 0 (1 9 9 8^7^)
1
Translation of WO 2004/033499
DESCRIPTION
CELL DEATH-INDUCING AGENT
5 The present invention relates to minibodies of antibodies that recognize HLA.
Background Art
The HLA class I antigen is formed by a heterodimer of a 45-KD a chain comprising
three domains (al, a2, a3), and a 12-KD P2 microglobulin. The main role of the HLA
10 molecule is to present CD8 + T cells with antigenic peptides, formed from about eight to ten
amino acids produced inside cells. As such, it plays a very important role in the immune
response and immune tolerance induced by this peptide presentation.
By ligating HLA class IA antigens with antibodies, cell growth-suppressing and cell
death-inducing effects have been observed in lymphocytes, suggesting that HLA molecules may
1 5 also be signal transduction molecules.
More specifically, for example, there are reports showing cell growth suppression of
activated lymphocytes by the B9.12.1 antibody against the al domain of human HLA class IA,
the W6/32 antibody against the a2 domain, and the TP25.99 and A1.4 antibodies against the a3
domain (non-patent literature 1, 2). Furthermore, two types of antibodies, MoAb90 and
20 YTH862, against the al domain have been reported to induce apoptosis in activated
lymphocytes (non-patent literature 2, 3, 4). Apoptosis induced by these two antibodies has
been shown to be a caspase-mediated reaction (non-patent literature 4), and therefore, HLA class
IA antigens expressed in lymphocytes are also speculated to be involved in apoptosis signal
transduction.
25 Furthermore, the 5H7 antibody against the a3 domain of human HLA class IA
(non-patent literature 5), and the RE2 antibody against the a2 domain of mouse HLA class IA
(non-patent literature 6) have been also reported to induce cell death in activated lymphocytes
and the like. However, in contrast with the aforementioned apoptosis-inducing antibodies
MoAb90 and YTH862, none of the cell deaths induced by these antibodies have been shown to
30 be mediated by caspase. Accordingly, cell deaths due to 5H7 and RE2 are predicted to be of a
type completely different from conventionally known apoptosis mechanisms.
As described above, there are numerous reports of the cell growth-suppressing actions
and cell death-inducing actions of anti-HLA antibodies. However, the antibodies used herein
are all in the molecular forms of IgG antibodies, F(ab')2, or Fab. To date there have been no
35 reports that cell death-inducing activity is enhanced by reducing the molecular weight of
antibodies, as in F(ab')2 and Fab.
2
Translation of WO 2004/033499
The 2D7 antibody is a mouse monoclonal antibody obtained by immunizing Balb/c
mice with human myeloma cells (non-patent literature 7). The 2D7 antibody has been observed
to bind very specifically to the cell surface of various lymphoid tumor cells, however, antigens
recognized by the 2D7 antibody have not been identified.
5 Prior art literature relating to the present invention of this application is shown below.
[Non-patent Document 1] Fayen et al, Int. Immunol. 10: 1347-1358(1998)
[Non-patent Document 2] Genestier et al., Blood 90: 3629-3639 (1997)
[Non-patent Document 3] Genestier et al., Blood 90: 726-735 (1997)
[Non-patent Document 4] Genestier et al. , J. Biol. Chem. 273 : 5060-5066 ( 1 998)
10 [Non-patent Document 5] Woodle et al., J. Immunol. 158: 2156-2164 (1997)
[Non-patent Document 6] Matsuoka et al.,}. Exp. Med. 181: 2007-20 1 5 ( 1 995 )
[Non-patent Document 7] Goto, et al. Blood 84: 1922 (1994)
Disclosure of the Invention
1 5 The primary purpose of this invention is to provide minibodies of antibodies that
recognize HLA class IA. A further objective of this invention is to provide novel therapeutic
agents for tumors or autoimmune diseases that utilize these minibodies.
To identify antigens of the 2D7 antibody, the present inventors used random hexamers
to synthesize cDNAs from the mRNAs purified from the 2D7 antigen-expressing cells,
20 RPMI8226. These were inserted into the retrovirus vector, pMX, and a retroviral expression
library was produced. The retrovirus expression library was packaged into a retrovirus by
transfection into BOSC23 cells. 2D7 antigens were screened by infecting NIH3T3 cells with
the virus thus obtained, staining these with 2D7 antibody, and then using FACS to perform
expression analysis. Cell lysates were then prepared from RPMI8226 cells and U266 cells
25 expressing the 2D7 antigen, and 2D7 antigens were identified by immunoprecipitation. As a
result of these examinations, 2D7 antigens were proven to be HLA class I molecules.
Since the molecules recognized by 2D7 antibodies are HLA class IA, the present
inventors examined whether 2D7 antibodies have cell death-inducing activity. More
specifically, Jurkat cells were cultured in the presence or absence of 2D7, with anti-mouse IgG
30 antibody also added. Cell nuclei were stained 48 hours later with Hoechst 33258, and then
checked for cell nuclei fragmentation, which is characteristic of dead cells. As a result, hardly
any cell death-inducing activity was observed in Jurkat cells with 2D7 antibody alone; however,
by further cross-linking the antibody with anti-mouse IgG antibody, nuclei fragmentation was
observed, a showing confirming that cell death was induced.
35 As described, because cross-linking with an anti-mouse IgG antibody is necessary for
2D7 antibody to induce cell death, it is difficult to clinically apply the 2D7 antibody to tumors or
3
Translation of WO 2004/033499
autoimmune diseases. Therefore, the present inventors examined the effect of reducing the
molecular weight of the 2D7 antibody on cell death induction. More specifically, genes
encoding the variable regions of the 2D7 antibody were cloned from hybridomas. The 2D7
antibody was then made into diabodies using genetic engineering techniques and the effects on
5 cell death-inducing activity was examined. Surprisingly, the 2D7 antibody converted to
diabodies showed strong cell death-inducing activity within a very short time and at low doses,
even without cross-linking with an anti-mouse IgG antibody. Furthermore, the diabody hardly
acted on normal peripheral blood-derived lymphocytes and adherent cells, and specifically
induced cell death in various myeloma cells, T cell leukemia cell lines, and activated
10 lymphocytes. The above-mentioned results show that the minibodies of antibodies recognizing
HLA can be utilized as cell death-inducing agents.
More specifically, the present invention provides the following [1] to [23]:
[I] a minibody that recognizes a human leukocyte antigen (HLA);
[2] the minibody of [1], wherein the HLA is an HLA class I;
1 5 [3] the minibody of [2], wherein the HLA class I is an HLA-A;
[4] a minibody derived from a 2D7 antibody;
[5] the minibody of any one of [1] to [4], wherein the minibody is a diabody;
[6] a minibody of any one of (a) to (d):
(a) a minibody comprising the amino acid sequence of SEQ ID NO: 6;
20 (b) a minibody functionally equivalent to the minibody of (a), and comprising an amino
acid sequence with a substitution, insertion, deletion and/or addition of one or more amino acids
in the amino acid sequence of SEQ ID NO: 6;
(c) a minibody comprising the amino acid sequences of CDRs of SEQ ID NOs: 2 and 4;
and
25 (d) a minibody functionally equivalent to the minibody of (c), and comprising an amino
acid sequence with a substitution, insertion, deletion and/or addition of one or more amino acids
in the amino acid sequence of the CDRs of SEQ ID NOs: 2 and 4;
[7] a method for producing an HLA-recognizing antibody having increased activity by
converting the HLA-recognizing antibody to a low-molecular-weight antibody;
30 [8] the method of [7], wherein the HLA is an HLA class I;
[9] the method of [8], wherein the HLA class I is an HLA-A;
[10] a method for producing a 2D7 antibody having increased activity by converting the 2D7
antibody to a low-molecular-weight antibody;
[II] the method of any one of [7] to [ 1 0] , wherein the conversion step comprises conversion to
35 a diabody;
[12] the method of any one of [7] to [1 1], wherein the activity is a cell death-inducing activity
4
Translation of WO 2004/033499
or a cell growth-suppressing activity;
[13] a cell death-inducing agent, comprising as an active ingredient the minibody of any one of
[1] to [6], the minibody produced by the method of any one of [7] to [12], or a 2D7 antibody;
[14] the cell death-inducing agent of [13] that induces cell death of a B cell or T cell;
5 [15] the cell death-inducing agent of [14], wherein the B cell or T cell is an activated B cell or
activated T cell;
[16] a cell growth-suppressing agent comprising as an active ingredient the minibody of any
one of [1] to [6], the minibody produced by the method of any one of [7] to [12], or a 2D7
antibody;
10 [17] an antitumor agent comprising as an active ingredient the minibody of any one of [ 1 ] to
[6], the minibody produced by the method of any one of [7] to [12], or a 2D7 antibody;
[ 1 8] the antitumor agent of [ 1 7], wherein the tumor is a blood tumor;
[19;] a therapeutic agent for an autoimmune disease, wherein the therapeutic agent comprises
as an active ingredient the minibody of any one of [1] to [6], the minibody produced by the
15 method of any one of [7] to [12], or a 2D7 antibody;
[20] the cell death-inducing agent of any one of [13] to [15], wherein the antibody is a diabody;
[21] the cell growth-suppressing agent of [16], wherein the antibody is a diabody;
[22] the antitumor agent of [ 1 7] or [ 1 8], wherein the antibody is a diabody; and
[23] the therapeutic agent for autoimmune disease of [19], wherein the antibody is a diabody;
20 The present invention provides minibodies that recognize HLA. The minibodies of
this invention are useful since their activity is elevated. Herein activity refers to a biological
action that is caused by binding an antibody to an antigen. Specific examples include cell
death-inducing actions, apoptosis-inducing actions, cell growth-suppressing actions, cell
differentiation-suppressing actions, cell division-suppressing actions, cell growth-inducing
25 actions, cell differentiation-inducing actions, cell division-inducing actions, and cell
cycle-regulating actions. Cell death-inducing actions and cell growth-suppressing actions are
preferred.
The cells that become the target of the above-mentioned actions, such as cell
death-inducing actions and cell growth-suppressing actions, are not particularly limited, though
30 hemocytes and suspended cells are preferred. Specific examples of hemocytes include
lymphocytes (B cells, T cells), neutrophils, eosinophils, basophils, monocytes (preferably
activated peripheral blood mononuclear cells (PBMC)), and myeloma cells, while lymphocytes
(B cells, T cells), and myeloma cells are preferred, and T cells or B cells (particularly activated B
cells or activated T cells) are most preferable. Suspended cells refer to cells that, when cultured,
35 grow in a suspended state without adhering to the surface of culturing vessels of glass, plastic or
the like. On the other hand, adherent cells refer to cells that, when cultured, adhere to the
5
Translation of WO 2004/033499
surface of culturing vessels of glass, plastic or the like.
In the present invention, administration of the minibodies that recognize HLA can treat
or prevent diseases such as tumors including blood tumors (hematopoietic tumors) (specific
examples include leukemia, myelodysplastic syndrome, malignant lymphoma, chronic
5 myelogenic leukemia, plasmacytic disorder (myeloma, multiple myeloma, macroglobulinemia),
and myeloproliferative disease (polycythemia vera, essential thrombocythemia, idiopathic
myelofibrosis)), and autoimmune diseases (specific examples include rheumatism, autoimmune
hepatitis, autoimmune thyroiditis, autoimmune bullosis, autoimmune adrenocortical disease,
autoimmune hemolytic anemia, autoimmune thrombycytopenic purpura, autoimmune atrophic
10 gastritis, autoimmune neutropenia, autoimmune orchitis, autoimmune encephalomyelitis,
autoimmune receptor disease, autoimmune infertility, Crohn's disease, systemic lupus
erythematosus, multiple sclerosis, Basedow's disease, juvenile diabetes, Addison's disease,
myasthenia gravis, lens-induced uveitis, psoriasis, and Behchet's disease).
In the present invention, HLA refers to human leukocyte antigen. HLA molecules are
15 categorized into class I and class II. Known examples of class I are HLA-A, B, C, E, F, G, H, J,
and such; and known examples of class II are HLA-DR, DQ, DP, and such. The antigens
recognized by the antibodies of this invention are not particularly limited, so long as they are
HLA molecules, preferably molecules classified as class I, and more preferably HLA-A.
In the present invention, a minibody comprises an antibody fragment that lacks a
20 portion of a whole antibody (for example, whole IgG). The minibodies of the present invention
are not particularly limited so long as they can bind an antigen. There are no particular
limitations on the antibody fragments of the present invention, so long as they are portions of a
whole antibody, and preferably contain a heavy chain variable region (VH) or a light chain
variable region (VL). More preferably, the antibody fragments contain both a heavy chain
25 variable region (VH) and a light chain variable region (VL). Specific examples of the antibody
fragments include Fab, Fab', F(ab')2, Fv, and scFv (single chain Fv), but are preferably scFv
(Huston, J. S. et ai, Proc. Natl. Acad. Sci. U.S.A. (1988) 85, 5879-5883; Plickthun "The
Pharmacology of Monoclonal Antibodies" Vol. 113, Resenburg and Moore Ed., Springer Verlag,
New York, pp. 269-3 1 5, (1 994)). Such antibody fragments can be prepared by treating an
30 antibody with an enzyme, such as papain or pepsin for example, to generate antibody fragments,
or by constructing genes that encode these antibody fragments, introducing them into expression
vectors, and then expressing them in appropriate host cells (see, for example, Co, M.S. et al.,
1994, J. Immunol. 152, 2968-2976; Better, M. and Horwitz, A. H., 1989, Methods Enzymol. 178,
476-496; Pluckthun, A. and Skerra, A., 1989, Methods Enzymol. 178, 497-515; Lamoyi, E.,
35 1986, Methods Enzymol. 121, 652-663; Rousseaux, J. et al, 1986, Methods Enzymol. 121,
663-669; Bird, R. E. and Walker, B. W., 1991, Trends Biotechnol. 9, 132-137).
6
Translation of WO 2004/033499
The minibodies of this invention preferably have smaller molecular weights than a
whole antibody, however, they may form multimers, including dimers, trimers, and tetramers,
and the molecular weights may become greater than that of the whole antibody.
A preferred minibody of this invention is an antibody comprising two or more antibody
5 VHs and two or more antibody VLs, in which each of these variable regions is linked directly or
indirectly via linkers and such. Such linkages may be covalent bonds or non-covalent bonds, or
may be both. An even more preferable minibody is an antibody comprising two or more
VH-VL pairs formed by non-covalent bonding between VH and VL. In this case, the distance
between one VH-VL pair and another VH-VL pair is preferably shorter in a minibody than in a
10 whole antibody.
A particularly favorable minibody of this invention is a diabody. A diabody is a dimer
formed by bonding two fragments, in which a variable region is linked to another variable region
via a linker and such (for example, scFv) (hereinafter referred to as diabody-constituting
fragments), and usually comprises two VLs and two VHs (P. Holliger et al, Proc. Natl. Acad. Sci.
15 USA, 90, 6444-6448 (1993); EP404097; W093/11161; Johnson et al, Method in Enzymology,
203, 88-98, (1991); Holliger et al, Protein Engineering, 9, 299-305, (1996); Perisic et al.,
Structure, 2, 1217-1226, (1994); John etal, Protein Engineering, 12(7), 597-604, (1999);
Holliger et al, Proc. Natl. Acad. Sci. USA., 90, 6444-6448, (1993); Atwell et al, Mol. Immunol.
33, 1301-1312, (1996)). The bonds between the diabody-constituting fragments may be
20 non-covalent or covalent bonds, but are preferably non-covalent bonds.
Alternatively, diabody-constituting fragments may be bound by a linker and such to
form a single chain diabody (sc diabody). In such cases, linking the diabody-constituting
fragments using a long linker of about 20 amino acids allows diabody-constituting fragments on
the same chain to form a dimer via non-covalent bonds to each other.
25 Diabody-constituting fragments include those with a linked VL-VH, linked VL-VL, and
linked VH-VH, and are preferably those with a linked VH-VL. In the diabody-constituting
fragments, the linker used to link a variable region to a variable region is not particularly limited,
but is preferably a linker short enough to prevent non-covalent bonding between variable regions
in the same fragment. The length of such a linker can be appropriately determined by those
30 skilled in the art, and is ordinarily 2 to 14 amino acids, preferably 3 to 9 amino acids, and most
preferably 4 to 6 amino acids. In this case, linkers between a VL and VH encoded on the same
fragment are short, and thus a VL and VH on the same strand do not form a non-covalent bond
nor a single-chain V region fragment, rather, the fragment forms a dimer with another fragment
via non-covalent bonding. Furthermore, according to the same principle as in diabody
35 construction, three or more diabody-constituting fragments may be bonded to form multimeric
antibodies, such as trimers and tetramers.
7
Translation of WO 2004/033499
Examples of the diabodies of this invention are, without limitation, a diabody
comprising the amino acid sequence of SEQ ID NO: 6, or a diabody that is functionally
equivalent to a diabody comprising the sequence of SEQ ID NO: 6, which comprises an amino
acid sequence with a mutation (substitution, deletion, insertion, and/or addition) of one or more
5 amino acids in the amino acid sequence of SEQ ID NO: 6; and a diabody comprising the amino
acid sequence of a complementarity-determining region (CDR) (or a variable region) of SEQ ID
NO: 2 and a CDR (or a variable region) of SEQ ID NO: 4, or a diabody that is functionally
equivalent to a diabody comprising the amino acid sequence of a CDR (or variable region) of
SEQ ID NO: 2 and a CDR (or a variable region) of SEQ ID NO: 4, which comprises an amino
10 acid sequence with mutations (substitution, deletion, insertion, and/or addition) of one or more
amino acids in the amino acid sequence of a CDR (or a variable region) of SEQ ID NO: 2 and a
CDR (or a variable region) of SEQ ID NO: 4.
Herein, "functionally equivalent" means that the diabody of interest has an activity
equivalent to an activity of a diabody comprising the sequence of SEQ ID NO: 6, or a diabody
15 comprising the sequence of a CDR (or a variable region) of SEQ ID NO: 2 and a CDR (or a
variable region) of SEQ ID NO: 4 (for example, HLA-A binding activity, and cell death-inducing
activity).
The number of mutated amino acids is not limited, but may ordinarily be 30 amino acids
or less, preferably 1 5 amino acids or less, and more preferably five amino acids or less (for
20 example, three amino acids or less).
Furthermore, a diabody comprising the amino acid sequence of SEQ ID NO: 6, or a
diabody comprising the sequence of a CDR (or a variable region) of SEQ ID NO: 2 and a CDR
(or a variable region) of SEQ ID NO: 4 may be humanized or chimerized to reduce heterologous
antigenicity against humans.
25 In the amino acid sequence of SEQ ID NO: 2, amino acids 1 to 134 correspond to the
variable region, amino acids 50 to 54 correspond to CDR1, amino acids 69 to 85 correspond to
CDR2, and amino acids 1 18 to 134 correspond to CDR3. In the amino acid sequence of SEQ
ID NO: 4, amino acids 1 to 128 correspond to the variable region, amino acids 46 to 55
correspond to CDR1, amino acids 71 to 77 correspond to CDR2, and amino acids 1 10 to 128
30 correspond to CDR3.
In the present invention, the HLA-recognizing minibodies specifically bind to HLA.
They are not particularly limited, so long as they have a biological action. The minibodies of
this invention can be prepared by methods well known to those skilled in the art. For example,
as described in the Examples, the antibodies can be prepared based on the sequence of an
35 HLA-recognizing antibody (particularly sequences of the variable regions and sequences of
CDRs), using genetic engineering techniques known to those skilled in the art.
Translation of WO 2004/033499
For the sequence of the HLA-recognizing antibody, a well-known antibody sequence
can be used, or an anti-HLA antibody can be prepared by a method well known to those skilled
in the art using HLA as the antigen, and then the sequence of this antibody can be obtained and
then used. Specifically, for example, this can be performed as follows: HLA protein or its
5 fragment is used as a sensitizing antigen to perform immunizations according to conventional
immunization methods, the obtained immunocytes are fused with well-known parent cells
according to conventional cell fusion methods, and monoclonal antibody-producing cells
(hybridomas) are then screened by ordinary screening methods. Antigens can be prepared by
known methods, such as a method using baculoviruses (W098/46777 and such). Hybridomas
1 0 can be prepared, for example, according to the method of Milstein et al. (Kohler, G. and Milstein,
C, Methods Enzymol. (1981) 73:3-46). When the antigen has low immunogenicity,
immunization can be performed using the antigen bound to immunogenic macromolecules, such
as albumin. Thereafter, cDNAs of the variable region (V region) of the antibody are
synthesized from the mRNAs of the hybridomas using reverse transcriptase, and the sequences
15 of the obtained cDNAs can be determined by known methods.
Antibodies that recognize HLA are not particularly limited, so long as they bind to
HLA; mouse antibodies, rat antibodies, rabbit antibodies, sheep antibodies, human antibodies,
and such may be used as necessary. Alternatively, artificially modified, genetically
recombinant antibodies, such as chimeric and humanized antibodies, may be used to reduce
20 heterologous antigenicity against humans. These modified antibodies can be produced using
known methods. A chimeric antibody is an antibody comprising the variable regions of the
heavy and light chains of an antibody from a non-human mammal such as a mouse, and the
constant regions of the heavy and light chains of a human antibody. The chimeric antibody can
be produced by linking a DNA encoding the variable regions of the mouse antibody with a DNA
25 encoding the constant regions of the human antibody, incorporating this into an expression vector,
and then introducing the vector to a host.
Humanized antibodies are also referred to as "reshaped human antibodies". Such
humanized antibodies are obtained by transferring the CDR of an antibody derived from a
non-human mammal, for example a mouse, to the CDR of a human antibody, and general gene
30 recombination procedures for this are also known. Specifically, a DNA sequence designed to
link a murine antibody CDR to the framework region (FR) of a human antibody can be
synthesized by PCR, using primers prepared from several oligonucleotides containing
overlapping portions of terminal regions. The obtained DNA is linked to a DNA encoding
human antibody constant regions, and this is then integrated into an expression vector, and the
35 antibody is produced by introducing this vector into host cells (see European Patent Application
EP 239400, and International Patent Application WO 96/02576). The human antibody FR to be
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linked via the CDR is selected so the CDR forms a favorable antigen-binding site. To form a
suitable antigen-binding site, amino acids in the framework region of the antibody variable
region may be substituted in the CDR of the reshaped human antibody, as necessary (Sato, K. et
al, 1993, Cancer Res. 53, 851-856).
5 These chimeric antibodies and humanized antibodies can be chimerized, humanized,
and such after their molecular weight is reduced, or their molecular weight can be reduced after
they have been chimerized, humanized, or such.
Methods for obtaining human antibodies are also known. For example, human
lymphocytes can be sensitized in vitro with a desired antigen, or with cells expressing the desired
10 antigen, and the sensitized lymphocytes can be fused with human myeloma cells, such as U266,
to obtain the desired human antibody with antigen-binding activity (Examined Published
Japanese Patent Application No. (JP-B) Hei 1 -59878). Further, a desired human antibody can
be obtained by using a desired antigen to immunize transgenic animals that have a full repertoire
of human antibody genes (see International Patent Application WO 93/12227, WO 92/03918,
15 WO 94/02602, WO 94/25585, WO 96/34096, and WO 96/33735). Furthermore, techniques for
obtaining human antibodies by panning using a human antibody library are also known. For
example, variable regions of human antibodies can be expressed as single chain antibodies
(scFvs) on the surface of phages using phage display methods, and phages that bind to antigens
can be selected. The DNA sequences that encode the variable regions of the human antibodies
20 binding the antigens can be determined by analyzing the genes of the selected phages. By
determining the DNA sequences of the scFvs that bind to the antigens, appropriate expression
vectors carrying relevant sequences can be produced to yield human antibodies. These methods
are already known, and are detailed in the following publications: WO 92/01047, WO 92/20791,
WO 93/06213, WO 93/11236, WO 93/19172, WO 95/01438, and WO 95/15388.
25 In the present invention, favorable examples of antibodies that recognize HLA include
2D7 antibodies. Examples of 2D7 antibodies are antibodies comprising the sequences of a
CDR (or a variable region) of SEQ ID NO: 2 and a CDR (or a variable region) of SEQ ID NO: 4,
but are not limited thereto. The 2D7 antibodies of this invention include an antibody which is
functionally equivalent to an antibody that comprises the sequence of a CDR (or a variable
30 region) of SEQ ID NO: 2 and a CDR (or a variable region) of SEQ ID NO: 4, and which
comprises an amino acid sequences with a mutation (substitution, deletion, insertion, and/or
addition) of one or more amino acids in the amino acid sequence of a CDR (or a variable region)
of SEQ ID NO: 2 and a CDR (or a variable region) of SEQ ID NO: 4. Herein, "functionally
equivalent" means that an antibody of interest has an activity (for example, HLA-A binding
35 activity, and cell death-inducing activity) equivalent to an antibody comprising the sequence of a
CDR (or a variable region) of SEQ ID NO: 2 and a CDR (or a variable region) of SEQ ID NO: 4.
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The number of mutated amino acids is not particularly limited, but may be ordinarily 30
amino acids or less, preferably 15 amino acids or less, and more preferably five amino acids or
less (for example, three amino acids or less). The amino acids are preferably mutated or
modified in a way that conserves the properties of the amino acid side chain. Examples of
5 amino acid side chain properties are: hydrophobic amino acids (A, I, L, M, F, P, W, Y, and V),
hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, and T), amino acids comprising the
following side chains: aliphatic side chains (G, A, V, L, I, and P); hydroxyl-containing side
chains (S, T, and Y); sulfur-containing side chains (C and M); carboxylic acid- and
amide-containing side chains (D, N, E, and Q); basic side chains (R, K, and H); aromatic
10 ring-containing side chains (H, F, Y, and W) (amino acids are represented by one-letter codes in
parentheses). Polypeptides comprising a modified amino acid sequence, in which one or more
amino acid residues is deleted, added, and/or replaced with other amino acids, are known to
retain their original biological activities (Mark, D. F. et al, Proc. Natl. Acad. Sci. USA 81,
5662-5666 (1984); Zoller, M. J. & Smith, M. Nucleic Acids Research 10, 6487-6500 (1982);
15 Wang, A. et al., Science 224, 143 1-1433; Dalbadie-McFarland, G. et al, Proc. Natl. Acad. Sci.
USA 79, 6409-6413 (1982)). In addition, the amino acid sequences of the antibody constant
regions and such are well known to those skilled in the art.
Furthermore, the 2D7 antibodies can be chimerized, humanized, or such by methods
well known to those skilled in the art. Such chimeric and humanized antibodies are also
20 included in the 2D7 antibodies of this invention.
The antibodies of this invention may be conjugated antibodies that are bonded to
various molecules, such as polyethylene glycol (PEG), radioactive substances, and toxins.
Such conjugate antibodies can be obtained by performing chemical modifications on the
obtained antibodies. Methods for antibody modification are established in this field. The term
25 "antibody" in this invention includes such conjugate antibodies.
The present invention includes DNAs that encode the antibodies of this invention.
This invention also includes DNAs encoding antibodies that hybridize under stringent conditions
to the aforementioned DNAs, and have antigen-binding capacity and activity. Hybridization
techniques (Sambrook, J. et al, Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab.
30 press, 1 989) are well known to those skilled in the art, and hybridization conditions can be
selected appropriately by those skilled in the art. Such hybridization conditions include, for
example, conditions of low stringency. Examples of conditions of low stringency include
post-hybridization washing in O.lx SSC and 0.1% SDS at 42°C, and preferably in O.lx SSC and
0.1% SDS at 50°C. More preferable hybridization conditions include those of high stringency.
35 Highly stringent conditions include, for example, washing in 5x SSC and 0.1% SDS at 65°C.
In these conditions, the higher the temperature, the higher the expectation of efficiently obtaining
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DNAs with a high homology. However, several factors, such as temperature and salt
concentration, can influence hybridization stringency, and those skilled in the art can suitably
select these factors to achieve similar stringencies.
The DNAs of this invention are used for in vivo and in vitro production of the antibodies
5 of this invention, and for other applications, such as gene therapy. The DNAs of this invention
may be in any form, so long as they encode the antibodies of this invention. More specifically,
they may be cDNAs synthesized from mRNAs, genomic DNAs, chemically synthesized DNAs,
or such. Furthermore, the DNAs of this invention include any nucleotide sequence based on the
degeneracy of the genetic code, so long as they encode the antibodies of this invention.
10 The antibodies of this invention can be produced by methods well known to those
skilled in the art. More specifically, a DNA of an antibody of interest is incorporated into an
expression vector. In so doing, the DNA is incorporated into the expression vector and
expressed under the control of an expression regulatory region such as an enhancer or promoter.
Next, antibodies can be expressed by transforming host cells with this expression vector. In this
15 regard, appropriate combinations of hosts and expression vectors can be used.
The vectors include, for example, Ml 3 vectors, pUC vectors, pBR322, pBluescript, and
pCR-Script. In addition to the above vectors, for example, pGEM-T, pDIRECT, and pT7 can
also be used for the subcloning and excision of cDNAs.
When using vectors to produce the antibodies of this invention, expression vectors are
20 particularly useful. When an expression vector is expressed in E. coli, for example, it should
have the above characteristics in order to be amplified in E. coli. Additionally, when E. coli
such as JM109, DH5 a , HB101, or XL 1 -Blue are used as the host cell, the vector preferably has
a promoter, for example, a lacZ promoter (Ward et al. (1989) Nature 341 : 544-546; (1992)
FASEB J. 6:2422-2427), araB promoter (Better et al. (1988) Science 240:1041-1043), or T7
25 promoter, to allow efficient expression of the desired gene in E. coli. Other examples of the
vectors include pGEX-5X-l (Pharmacia), "QIAexpress system" (QIAGEN), pEGFP, and pET
(where BL21, a strain expressing T7 RNA polymerase, is preferably used as the host).
Furthermore, the vector may comprise a signal sequence for polypeptide secretion.
When producing proteins into the periplasm of E. coli, the pelB signal sequence (Lei, S. P. et al.
30 J. Bacterid. 169:4379 (1987)) may be used as a signal sequence for protein secretion. For
example, calcium chloride methods or electroporation methods may be used to introduce the
vector into a host cell.
In addition to E. coli, expression vectors derived from mammals (e.g., pCDNA3
(Invitrogen), pEGF-BOS (Nucleic Acids Res. (1990) 18(17):5322), pEF, pCDM8), insect cells
35 (e.g., "Bac-to-BAC baculovirus expression system" (GIBCO-BRL), pBacPAK8), plants (e.g.,
pMHl, pMH2), animal viruses (e.g., pHSV, pMV, pAdexLcw), retroviruses (e.g., pZIPneo),
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yeasts (e.g., "Pichia Expression Kit" (Invitrogen), pNVl 1, SP-Q01), and Bacillus subtilis (e.g.,
pPL608, pKTH50) may also be used as a vector for producing the polypeptide of the present
invention.
In order to express proteins in animal cells, such as CHO, COS, and NIH3T3 cells, the
5 vector preferably has a promoter necessary for expression in such cells, for example, an SV40
promoter (Mulligan et al. (1979) Nature 277:108), MMLV-LTR promoter, EFlapromoter
(Mizushima et al. (1990) Nucleic Acids Res. 18:5322), CMV promoter, etc.). It is even more
preferable that the vector also carry a marker gene for selecting transformants (for example, a
drug-resistance gene enabling selection by a drug, such as neomycin and G418). Examples of
1 0 vectors with such characteristics include pMAM, pDR2, pBK-RS V, pBK-CMV, pOPRSV,
pOP 13, and such.
In addition, to stably express a gene and amplify the gene copy number in cells, CHO
cells having a defective nucleic acid synthesis pathway can be introduced with a vector
containing a DHFR gene (for example, pCHOI) to compensate for the defect, and the copy
15 number may be amplified using methotrexate (MTX). Alternatively, a COS cell, which carries
an SV40 T antigen-expressing gene on its chromosome, can be transformed with a vector
containing the SV40 replication origin (for example, pcD) for transient gene expression. The
replication origin may be derived from polyoma viruses, adenoviruses, bovine papilloma viruses
(BPV), and such. Furthermore, to increase the gene copy number in host cells, the expression
20 vector may contain, as a selection marker, an aminoglycoside transferase (APH) gene, thymidine
kinase (TK) gene, E. coli xanthine guanine phosphoribosyl transferase (Ecogpt) gene,
dihydrofolate reductase (dhfr) gene, and such.
Methods for expressing the DNAs of this invention in the bodies of animals include
methods of incorporating the DNAs of this invention into appropriate vectors and introducing
25 them into living bodies by, for example, a retrovirus method, liposome method, cationic
liposome method, or adenovirus method. The vectors that are used include adenovirus vectors
(for example, pAdexlcw), and retrovirus vectors (for example, pZIPneo), but are not limited
thereto. General genetic manipulations such inserting the DNAs of this invention into vectors
can be performed according to conventional methods (Molecular Cloning, 5.61-5.63).
30 Administration to living bodies can be carried out by ex vivo method or in vivo methods.
Furthermore, the present invention provides host cells into which a vector of this
invention is introduced. The host cells into which a vector of this invention is introduced are
not particularly limited; for example, E. coli and various animal cells are available for this
purpose. The host cells of this invention may be used, for example, as production systems to
35 produce and express the antibodies of the present invention. In vitro and in vivo production
systems are available for polypeptide production systems. Production systems that use
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eukaryotic cells or prokaryotic cells are examples of in vitro production systems.
Eukaryotic cells that can be used include, for example, animal cells, plant cells, and
fungal cells. Known animal cells include: mammalian cells, for example, CHO (J. Exp. Med.
(1995)108, 945), COS, 3T3, myeloma, BHK (baby hamster kidney), HeLa, Vero, amphibian
5 cells such asXenopus laevis oocytes (Valle, et al. (1981) Nature 291, 358-340), or insect cells
(e.g., Sf9, Sf21, and Tn5). CHO cells in which the DHFR gene has been deleted, such as
dhfr-CHO (Proc. Natl. Acad. Sci. USA (1980) 77, 4216-4220) and CHO K-l (Proc. Natl. Acad.
Sci. USA (1968) 60, 1275), are particularly preferable for use as CHO cells. Of the animal cells,
CHO cells are particularly favorable for large-scale expression. Vectors can be introduced into
10 a host cell by, for example, calcium phosphate methods, DEAE-dextran methods, methods using
cationic liposome DOTAP (Boehringer-Mannheim), electroporation methods, lipofection
methods, etc.
Plant cells include, for example, Nicotiana tabacum-derived cells known as polypeptide
production systems. Calluses may be cultured from these cells. Known fungal cells include
1 5 yeast cells, for example, the genus Saccharomyces, such as Saccharomyces cerevisiae; and
filamentous fungi, for example, the genus Aspergillus such as Aspergillus niger.
Bacterial cells can be used in prokaryotic production systems. Examples of bacterial
cells include E. coli (for example, JM109, DH5a, HB101 and such); and Bacillus subtilis.
Antibodies can be obtained by transforming the cells with a polynucleotide of interest,
20 then culturing these transformants in vitro. Transformants can be cultured using known
methods. For example, DMEM, MEM, RPMI 1640, or IMDM may be used as the culture
medium for animal cells, and may be used with or without serum supplements such as fetal calf
serum (FCS). Serum-free cultures are also acceptable. The preferred pH is about 6 to 8 over
the course of culturing. Incubation is typically carried out at a temperature of about 30 to 40°C
25 for about 1 5 to 200 hours. Medium is exchanged, aerated, or agitated, as necessary.
On the other hand, production systems using animal or plant hosts may be used as
systems for producing polypeptides in vivo. For example, a DNA of interest may be introduced
into an animal or plant, and the polypeptide produced in the body of the animal or plant is then
recovered. The "hosts" of the present invention include such animals and plants.
30 When using animals, there are production systems using mammals or insects.
Mammals such as goats, pigs, sheep, mice, and cattle may be used (Vicki Glaser SPECTRUM
Biotechnology Applications (1993)). Alternatively, the mammals may be transgenic animals.
For example, a DNA of interest may be prepared as a fusion gene with a gene encoding
a polypeptide specifically produced in milk, such as the goat P-casein gene. DNA fragments
35 containing the fusion gene are injected into goat embryos, which are then introduced back to
female goats. The desired antibody can then be obtained from milk produced by the transgenic
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goats, which are born from the goats that received the embryos, or from their offspring.
Appropriate hormones may be administered to increase the volume of milk containing the
polypeptide produced by the transgenic goats (Ebert, K.M. et al, Bio/Technology 12, 699-702
(1994)).
5 Insects, such as silkworms, may also be used. Baculoviruses carrying a DNA of
interest can be used to infect silkworms, and the antibody of interest can be obtained from their
body fluids (Susumu, M. et al, Nature 315, 592-594 (1985)).
When using plants, tobacco can be used, for example. When tobacco is used, a DNA
of interest may be inserted into a plant expression vector, for example, pMON 530, and then the
10 vector may be introduced into a bacterium, such as Agrobacterium tumefaciens. The bacteria
are then used to infect tobacco, such as Nicotiana tabacum, and the desired polypeptides are
recovered from the leaves (Julian K.-C. Ma et al, Eur. J. Immunol. 24, 131-138 (1994)).
The resulting antibodies of this invention may be isolated from the inside or outside
(such as the medium) of host cells, and purified as substantially pure and homogenous antibodies.
1 5 Any standard method for isolating and purifying antibodies may be used, and methods are not
limited to any specific method. Antibodies may be isolated and purified by selecting an
appropriate combination of, for example, chromatographic columns, filtration, ultrafiltration,
salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation,
SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, recrystallization, and
20 others.
Chromatography includes, for example, affinity chromatography, ion exchange
chromatography, hydrophobic chromatography, gel filtration, reverse-phase chromatography, and
adsorption chromatography (Strategies for Protein Purification and Characterization: A
Laboratory Course Manual. Ed Daniel R. Marshak et al, Cold Spring Harbor Laboratory Press,
25 1 996). These chromatographies can be carried out using liquid phase chromatographies such as
HPLC and FPLC. The present invention also includes antibodies that are highly purified using
these purification methods.
In the present invention, the antigen-binding activity of antibodies (Antibodies A
Laboratory Manual. Ed Harlow, David Lane, Cold Spring Harbor Laboratory, 1988) can be
30 measured using well known techniques. For example, ELISA (enzyme linked immunosorbent
assay), EIA (enzyme immunoassay), RIA (radioimmunoassay), or fluoroimmunoassay may be
used.
In the present invention, whether or not the antibodies of this invention induce cell death
in suspended cells can be determined from whether cell death is induced in Jurkat cells or
35 ARH77 cells, as in the Examples. Whether or not the antibodies induce cell death in adhesion
cells can be determined from whether cell death is induced in HeLa cells, as in the Examples.
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Furthermore, the present invention provides cell death-inducing agents or cell
growth-suppressing agents which comprise minibodies or 2D7 antibodies of this invention as
active ingredients. The cell death-inducing activity of the minibodies or 2D7 antibodies in this
invention is considered to have a particularly large effect on activated T cells or B cells, therefore,
5 it is considered to be particularly effective for treatment and prevention of tumors such as cancer
(particularly blood tumors), and autoimmune diseases. Accordingly, the present invention
provides methods of treatment and prevention of tumors such as cancer (particularly blood
tumors), and autoimmune diseases that use the minibodies or 2D7 antibodies of this invention.
When using 2D7 antibodies whose molecular weight has not been reduced as active ingredients,
10 they are preferably cross-linked with an anti-IgG antibody and such.
The above-mentioned antibodies can also be used as conjugate antibodies, after linking
to various reagents. Examples of such reagents include chemotherapy reagents, radioactive
substances, and toxins. Such conjugate antibodies can be produced by known methods
(US50573 1 3, and US5 1 56840).
1 5 The above-mentioned pharmaceutical agents can be directly administered to patients, or
administered as pharmaceutical compositions formulated by known pharmaceutical methods.
For example, they may be administered orally, as tablets, capsules, elixirs, or microcapsules,
sugar-coated as necessary; or parenterally, in the form of injections of sterile solution or
suspensions prepared with water or other pharmaceutically acceptable liquids. For example,
20 they may be formulated by appropriately combining them with pharmaceutically acceptable
carriers or media, more specifically, sterilized water or physiological saline solutions, vegetable
oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, vehicles,
preservatives, binding agents, and such, and mixing them at a unit dosage form required for
generally accepted pharmaceutical practice. The amount of active ingredient in the formulation
25 is such that appropriate doses within indicated ranges are achieved.
Additives that can be mixed into tablets and capsules include, for example, binding
agents such as gelatin, cornstarch, tragacanth gum, and gum arabic; excipients such as crystalline
cellulose; swelling agents such as cornstarch, gelatin, alginic acid; lubricants such as magnesium
stearate; sweeteners such as sucrose, lactose, or saccharine; and flavoring agents such as
30 peppermint and Gaultheria adenothrix oils, or cherry. When the unit dosage form is a capsule,
liquid carriers, such as oils and fats, can be further included in the above-indicated materials.
Sterile compositions to be injected can be formulated using a vehicle such as distilled water used
for injection, according to standard protocols.
Aqueous solutions used for injections include, for example, physiological saline and
35 isotonic solutions comprising glucose or other adjunctive agents such as D-sorbitol, D-mannose,
D-mannitol, and sodium chloride. They may also be combined with appropriate solubilizing
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agents, such as alcohol, and specifically, ethanol, polyalcohol such as propylene glycol or
polyethylene glycol, or non-ionic detergent such as polysorbate 80™ or HCO-50, as necessary.
Oil solutions include sesame oils and soybean oils, and can be combined with
solubilizing agents such as benzyl benzoate or benzyl alcohol. Injection solutions may also be
5 formulated with buffers, for example, phosphate buffers or sodium acetate buffers; analgesics,
for example, procaine hydrochloride; stabilizers, for example, benzyl alcohol or phenol; or
anti-oxidants. The prepared injections are typically aliquoted into appropriate ampules.
Administration to patients may be performed, for example by intra-arterial injection,
intravenous injection, or subcutaneous injection, alternatively by intranasal, transbronchial,
10 intramuscular, transdermal, or oral administration using methods well known to those skilled in
the art. Doses vary depending on the body weight and age of the patient, method of
administration and such; nevertheless, those skilled in the art can appropriately select suitable
doses. Furthermore, if a compound can be encoded by a DNA, the DNA may be incorporated
into a gene therapy vector to carry out gene therapy. Doses and administration methods vary
15 depending on the body weight, age, and symptoms of patients, but, again, they can be
appropriately selected by those skilled in the art.
A single dose of a pharmaceutical agent of this invention varies depending on the target
of administration, the target organ, symptoms, and administration method. However, an
ordinary adult dose (presuming a body weight of 60 kg) in the form of an injection is
20 approximately 0.1 to 1000 mg, preferably approximately 1 .0 to 50 mg, and more preferably
approximately 1 .0 to 20 mg per day, for example.
When administered parenterally, a single dose varies depending on the target of
administration, the target organ, symptoms, and administration method, but in the form of an
injection, for example, a single dose of approximately 0.01 to 30 mg, preferably approximately
25 0. 1 to 20 mg, and more preferably approximately 0. 1 to 1 0 mg per day may be advantageously
administered intravenously to an ordinary adult (presuming a body weight of 60 kg). For other
animals, a converted amount based on the amount for a body weight of 60 kg, or a converted
amount based on the amount for a body surface area can be administered.
30 Brief Description of the Drawings
Fig. 1 shows the adaptors used to produce the pMX2 vector. The bold letters indicate
BstXI recognition sequences.
Fig. 2A and Fig. 2B show 2D7 antigen expression in cell lines. Each cell type was
stained with 2D7 antibody and their expressions were examined. (Solid line: no primary
35 antibody; dotted line: 2D7 antibody)
Fig. 3 is a set of photographs showing the results of immunoprecipitation using the 2D7
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antibody. NIH3T3, RPMI8226, and U266 cells were solubilized, immunoprecipitation was
performed with the 2D7 antibody, anti-BST-1 antibody (control), or protein G itself, and the
proteins were detected by silver staining. In RPMI8226 and U266, a molecule of
approximately 12 KD (arrow), which is specifically precipitated by the 2D7 antibody, is detected.
5 This band was cut out and peptide sequenced, and thus found to be p2-microglobulin.
Fig. 4 shows flow diagrams for screening. Separation into pools, preparation of DNA,
packaging into virus, infection of 3T3 cells, and screening using FACS were performed in one
span (Fig. 4A). By the end of the fourth screening, the library was narrowed down to
approximately 20 clones. In the fifth screening, 64 colonies were individually inoculated into a
10 96-well plate, pools were formed using the vertical and horizontal rows, and then screened. As
a result, the library was narrowed down to twelve candidate clones (Fig. 4B).
Fig. 5 shows the results of screening using FACS. Fig. 5A shows the results of the
second screening, Fig. 5B shows the results of the third screening, and Fig. 5C shows the results
of the fourth screening. NIH3T3 cells were infected with retroviruses prepared from each pool,
15 and three days later the cells were stained with the 2D7 antibody. The clones were narrowed
down by gradually reducing the pool size of each screening.
Fig. 6 shows the results of screening using FACS. Fig. 6A shows the results of the
fifth screening, and Fig. 6B shows the result of the final screening. As a result of the fifth
screening, positive clones were found in rows 3, 4, 6, and 8, and in rows E, F, and G. As a
20 result of screening the twelve candidate clones, positive clones were found in row E at 6E .
When the nucleotide sequence of this 6E was analyzed, it was found to encode HLA classl
A*6802.
Fig. 7 is a graph and a set of photographs showing the influence on cells of the addition
of 2D7 antibody. 2D7 antibody (10 ug/ml) was added, and the number of viable cells was
25 determined 48 hours later. Hardly any change in cell growth was observed, even after 2D7
antibody was added (Fig. 7A). K562 cells (Fig. 7B), Jurkat cells (Fig. 7C), and RPMI8226
cells (Fig. 7D) were each observed 24 hours after antibody addition. The 2D7 antibody induced
aggregation of Jurkat cells.
Fig. 8 is a set of photographs showing cell death induction due to cross-linking of the
30 2D7 antibody. Each combination of the 2D7 antibody with anti-mouse IgG was made to act on
Jurkat cells, and the cell nuclei were stained 48 hours later. Nuclear fragmentation due to cell
death was observed when the 2D7 antibody and anti-mouse IgG acted on cells simultaneously.
Fig. 9 shows a 2D7 diabody (2D7DB) sequence.
Fig. 10A and Fig. 10B show a 2D7 diabody structure. Fig. 10C is a photograph
35 showing its transient expression in COS7 cells.
Fig. 1 1 A and Fig. 1 IB show the cytotoxic activity of 2D7DB transiently expressed in
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COS7.
Fig. 12 shows the cytotoxic activity of 2D7DB transiently expressed in COS7. K562
cells (Fig. 12 A) and Jurkat cells (Fig. 12B) were used.
Fig. 13 shows the cytotoxic activity of 2D7DB transiently expressed in COS7.
5 RPMI8226 cells (Fig. 1 3 A), IL-KM3 cells (Fig. 1 3B), U266 cells (Fig. 1 3C), and ARH77 cells
(Fig. 1 3D) were used.
Fig. 14 is a graph showing the growth-suppressing effect of purified 2D7DB.
Fig. 15 shows cell death induction by purified 2D7DB, 48 hours after induction.
ARH77 cells (Fig. 15 A), Jurkat cells (Fig. 15B), K562 cells (Fig. 15C), and HeLa cells (Fig.
10 15D) were used.
Fig. 16 shows cell death induction by purified 2D7DB, 48 hours after induction. U266
cells (Fig. 16A), and IL-KM3 cells (Fig. 16B) were used for the study.
Fig. 17 shows a time course of cell death induction by 2D7DB (2 ug/ml). Cell death
induction was investigated at 12 through to 38 hours. ARH77 cells (Fig. 1 7A) and Jurkat cells
15 (Fig. 17B) were used.
Fig. 18 shows a time course of cell death induction by 2D7DB (2 ug/ml). Cell death
induction was investigated at three through to six hours. ARH77 cells (Fig. 18A) and Jurkat
cells (Fig. 18B) were used.
Fig. 19 shows the effect of Z-VAD-FMK on cell death due to 2D7DB. The study was
20 performed using ARH77 cells 1 6 hours after induction.
Fig. 20 shows the effect of Z-VAD-FMK on cell death due to 2D7DB. The study was
performed using Jurkat cells 16 hours after induction.
Fig. 2 1 is a set of photographs showing that cell death due to 2D7DB is not
accompanied by DNA fragmentation. The study was performed 24 hours after cell death
25 induction.
Fig. 22 shows the results of investigating the effect of cytochalasin D on the cell
death-inducing activity of 2D7DB. By pre-treating ARH77 cells with cytochalasin D, which is
an actin-polymerization inhibitor, the cells showed resistance to 2D7DB-induced cell death.
Fig. 23 is a set of photographs showing the results of immunostaining to investigate the
30 state of the intracellular actin and nuclei. After reacting ARH77 cells under the conditions
described in the figure, actin was detected using anti-actin antibody (red), and cell nuclei were
detected using Hoechst 33258 (blue). Actin was absent from cells treated with 2D7DB.
Fig. 24 shows that the 2D7DB suppresses an increase in human IgG (hlgG)
concentration in serum in a mouse model of human myeloma. The data shows the average +
35 SEM. There was a significant difference (*: p<0.05) between the vehicle-administered group
and the 2D7DB-administered group, according to unpaired t-tests.
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Fig. 25 shows that the 2D7DB has a life-prolonging effect in a mouse model of human
myeloma. There was a significant difference (*: p<0.05) between the vehicle-administered
group and the 2D7DB-administered group, according to generalized Wilcoxon tests.
Fig. 26 shows analyses of the action of 2D7DB on PBMC. PHA-M (Fig. 26A), ConA
5 (Fig. 26B), and SAC (Fig. 26C) were used as mitogens. Fig. 26D shows the results in the
absence of a mitogen, and Fig. 26E shows the results of a positive control (ARH77). The
results shown are, from the top, those of no 2D7DB addition, three-hour addition, and 24-hour
addition.
10 Best Mode for Carrying out the Invention
Herein below, the present invention is specifically described using Examples; however,
it should not to be construed as being limited thereto.
[1] Cell lines
15 Human myeloma cell lines (RPMI8226, K562, and ARH77), human T-cell leukemia cell
line (Jurkat), FDC-P 1 , HCI- 1 6, and 2D7 hybridoma cell line (from University of Tokushima)
were cultured in RPMI1640 medium (GIBCO BRL) supplemented with 10% fetal calf serum
(FCS). Human myeloma cell lines (IL-KM3 and U266) were individually cultured in the same
medium supplemented with 2 ng/ml of IL-6 (R & D), and Ba/F3 was cultured in the same
20 medium supplemented with 2 ng/ml of IL-3 (R & D). COS7, 293T, HeLa, NIH3T3, and
BOSC23 were cultured in DMEM medium (GIBCO BRL) supplemented with 10% FCS, and
CHO was cultured in a-MEM medium (GIBCO BRL) supplemented with 5% FCS or 10% FCS.
[2] Production of pMX2 vectors
25 The GFP gene region of the retrovirus vector, pMX-GFP, which packages the GFP gene
in the virus particle, was cut out and removed using EcoRI-Sall. The adaptor, which comprised
a BstXI site in its sequence (Fig. 1) (and was synthesized with an ABI DNA synthesizer, then
annealed in vitro before use), was inserted into this region, forming pMX2.
30 [3] Production of cDNA libraries
Total RNA was purified from RPMI8226 cells by standard methods using Trisol
(GIBCO BRL). Furthermore, the mRNAs were purified from 200 ug of this total RNA, using a
uMACS mRNA Isolation kit (Miltenyi Biotec) according to the manufacturer's instructions.
The cDNAs were synthesized using random hexamers (Superscript Choice System for cDNA
35 Synthesis; Invitrogen) with 3.6 ug of mRNA as template, and then a BstXI adaptor (Invitrogen)
was linked to both ends. This cDNA was inserted into a pMX2 vector cleaved with BstXI, and
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was introduced into ELECTRO MAX DH10B (GIBCO BRL) by electroporation (2.5 KV, 200 fi,
25 uF). After adding 1 ml of SOC, the vectors were then incubated at 37°C for one hour, 1 ml
of 40% glycerol/LB+Amp was added. A portion of the culture was used to check the titer and
the remainder was stored at -80°C. The obtained library was plated at 200 ul/well (7%
5 DMSO/LB+Amp) into two 96-well plates, so that each well contained 1000 clones. These
were cultured overnight at 37°C. Four wells (4000 clones) from this plate were combined and
placed into an ampicillin-containing LB medium (4 ml). This was defined as one pool, the rest
of the wells were treated similarly. Ultimately, 24 pools were prepared from a single plate.
After incubating each pool overnight at 37°C, DNAs were prepared (QIAGEN) and used for
10 transfection into packaging cells. The plates used for inoculation were stored at -80°C until
used for secondary screening.
[4] Purification of antibodies
0.5 ml of ascites, sent from University of Tokushima, was adsorbed to a Protein A Hi
15 Trap Affinity column (Amersham Pharmacia). The IgG fraction was then eluted using 0.1 M
sodium citrate, pH3.0, and the 2D7 antibody was collected. This was concentrated using
Centricon (YM-10; Millipore), and the buffer was exchanged to PBS to ultimately yield a total of
5.34 mg of antibody. This was separated into aliquots and stored at -20°C (concentration: 0.89
ug/uL).
20
[5] FACS
Adherent cells were detached using 1 mM EDTA/PBS, and suspended cells were
collected by centrifugation, then suspended in FACS buffer (2.5% FCS, 0.02% NaN 3 /PBS).
These cells were left to stand on ice for one hour in a buffer (5% FCS/PBS) containing 2D7
25 antibody (final concentration 10 |ig/ml). These were then washed with FACS buffer, reacted in
a solution of FITC -anti-mouse IgG (Immunotech) (1 : 150, 50 uL FACS buffer) on ice for 30
minutes, washed twice with FACS buffer, and then analyzed using EPICS ELITE (COULTER).
[6] Retrovirus infection
30 (i) Retrovirus packaging
The day before transfection, 2ml of BOSC23 cells, which are retrovirus-packaging cells,
were plated onto a 6-well plate at 6 x 10 5 cells/well. Transfection was carried out by the
following procedure: 1 ug of the plasmid DNA derived from each pool was mixed with 3 uL of
FuGENE 6 Transfection Reagent (Roche), left to stand at room temperature for 20 minutes, and
35 then added to the BOSC23 cell culture medium plated the day before. Cells were then cultured
at 37°C for 48 hours, and the culture medium was collected. Dead cells were removed by
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centrifugation at 3000 rpm for five minutes, and the culture solution was then used as the virus
solution.
(ii) Virus infection
The 2 ml of NIH3T3 cells plated onto 6-well plates at 1 x 10 5 cells/well the day before
5 were cultured for 24 hours in 1 ml of virus solution supplemented with 10 u^g/ml of polybrene
(hexadimethrine bromide; Sigma). 1.5 ml of fresh medium was then added, the cells were
cultured for another 48 hours, and gene expression was then analyzed using FACS.
[7] Immunoprecipitation
10 Cells were lysed in a lysis buffer (0.5% Nonidet P-40, 10 mM Tris, pH 7.6, 150 mM
NaCl, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 5 fig/ml aprotinin), and the resulting
solution was centrifuged to remove the insoluble proteins and obtain a cell lysate. 1 ug of 2D7
antibody was added, and incubated at 4°C for four hours. Magnetic protein G (BioMag) was
then added, and this was incubated for another one hour. Subsequently, the immunoconjugate
15 was washed three times with a lysis buffer, and then subjected to SDS-PAGE. This gel was
silver stained (Daiichi Pure Chemicals) according to the attached instructions. On the other
hand, for peptide sequencing, the gel on which SDS-PAGE was performed was transferred to
ProBlott (Applied Biosystems), and this was stained for one minute with Coomassie blue
staining solution (0. 1% coomassie blue R-250 in 40% MetOH/ 1% acetic acid). After washing
20 several times with 50% MetOH, the band of interest was cut out, washed five times with 1 ml of
DDW, dried in vacuo, and then subjected to peptide sequencing.
[8] Cell growth assay using the 2D7 antibody
Each type of cell was plated into a 96-well plate at 1 x 10 6 cells/ml in the presence or
25 absence of PMA (50 ng/ml; GIBCO BRL) and PHA (10 ul/ml; GIBCO BRL). After
subsequent addition (10 ng/ml) or no addition of the 2D7 antibody, this was cultured for 48
hours. After culturing, morphological changes in the cells were observed under a microscope.
Viable cell count was determined by adding WST-8 (viable cell count reagent SF; Nacalai
Tesque), culturing at 37°C for two hours, and measuring OD450 to measure the relative viable cell
30 count.
[9] Induction of cell death by cross- linking
Jurkat cells were plated on a 24-well plate at 8 x 10 5 cells/well, and 10 (xg/ml of
anti-mouse IgG (Fc) antibody (Cappel) was further added in the presence (5 ug/ml) or absence
35 of 2D7 antibody. 48 hours later, the cells were collected, and after washing with PBS, methanol
was added to a concentration of 70%, and this was left to stand at -20°C for 1 5 minutes. After
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washing the cells with FACS buffer several times, Hoechst 33258 was added at a concentration
of 10 (ag/ml, and this was incubated at room temperature for 30 minutes. The cells were
washed again with FACS Buffer, and then placed on a slide glass as a droplet to observe the state
of the nuclei under a fluorescence microscope.
5
[10] Cloning of the 2D7 variable region
Total RNA was purified from 2D7 hybridoma (provided from University of Tokushima)
using Trizol according to standard methods. Using 3 ug of this RNA as a template, cDNAs
were synthesized using a SMART RACE cDNA Amplification kit (CLONTECH), according to
10 the attached instructions. Using this cDNA as a template, the variable regions of the heavy
chain and light chain were amplified by PCR using the following primers:
Heavy chain: 5'-CAGGGGCCAGTGGATAGACTGATG (SEQ ID NO: 9)
Light chain: 5 '-GCTCACTGGATGGTGGGAAGATG (SEQ ID NO: 10)
The amplified cDNAs encoding each of variable regions were subcloned into pCR-TOPO vector
15 (Invitrogen), and the nucleotide sequences (SEQ ID NOs: 1 and 3) were determined.
[11] Production of 2D 7 diabody expression vector
Plasmids, to which each of the variable region cDNAs were subcloned, were used as
templates, and the variable regions of the heavy chain and light chain (VH and VL) were
20 respective amplified using the primers below:
Heavy chain
2D7DB-H1: 5'-CCTGAATTCCACCATGCGATGGAGCTGGATCTTTC (SEQ ID NO: 11)
2D7DB-H2: 5 '-AATTTGGCTACCGCCTCCACCTGAGGAGACTGTGAGAGTGGTGCCCT
(SEQ ID NO: 12)
25 Light chain
2D7DB-L1 : 5 ' -TCCTCAGGTGG AGGCGGTAGCCAAATTGTTCTCACCCAGTCGCCAGC
(SEQ ID NO: 13)
2D7DB-L2:
5'-ATTGCGGCCGCTTATCACTTATCGTCGTCATCCTTGTAGTCTTTTATCTCCAACTTTG
30 TCCCCGAGCC (SEQ ID NO: 14)
Each of the VH and VL cDNAs amplified by these primers were combined into one
tube, and further subjected to PCR. Using the PCR products as templates, PCR was performed
again, this time using 2D7DB-H1 and 2D7DB-L2 as primers, to synthesize cDNA with VH and
VL linked through a 5-mer linker (SEQ ID NO: 5). This cDNA was digested with EcoRI-NotI
35 and inserted into the EcoRI-NotI gap of the animal cell expression vector, pCXND3. The
nucleotide sequence was confirmed, completing the construction of the 2D7 diabody expression
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vector, pCXND3-2D7DB.
[12] Transient expression in COS7 cells
2 ug of pCXND3-2D7DB, or of an empty vector used as a control, was mixed with 6 uL
5 of transfection reagent (LT-1, MIRUS) according to the attached instructions, and this was added
to COS7 cells (plated the day before into a 6-well plate at lx 10 5 cells/well) whose medium had
been exchanged to a serum-free medium (OPTI-MEM, GIBCO BRL). Five hours later, 200 uL
of serum was added, and this was cultured for two to three days. The medium was collected,
and dead cells were removed by centrifugation. The culture supernatant was then used for an
10 experiment to detect cytotoxic activity.
Expression of 2D7DB in the culture supernatant was confirmed by Western blotting.
More specifically, equal amounts of 2x SDS-PAGE Sample buffer and culture supernatant were
added. In addition, after lysing the cells by adding a lysis buffer (0.5% Nonidet P-40, 10 mM
Tris, pH 7.6, 150 mM NaCl, 5 mM EDTA), insolubilized proteins were removed by
1 5 centrifugation to prepare a cell lysate, and an equal amount of 2x SDS-PAGE Sample buffer was
added to this. After performing SDS-PAGE on each sample, the gels were transferred to PVDF
membranes, and expression of the 2D7 single chain was detected using anti-FLAG antibody.
[13] Establishment of expression cell lines producing 2D7 diabody
20 20 ug of pCXND3-2D7DB, linearized by cleaving with Pvul, was introduced to CHO
cells (DXB 1 1 strain) by electroporation, as described below.
After washing the CHO cells twice with ice-cold PBS, they were suspended in PBS at
lx 10 7 cells/ml. 20 ug of the above-mentioned plasmid was mixed into these cells, and this was
electropulsed (1 .5 KV, 25 uFD). The cells were diluted in to appropriate fractions, plated on to
25 a 10 cm dish, and cultured in the presence of G418 (GIBCO BRL) at a final concentration of 50
pg/ml. Approximately 30 clones were selected from the grown colonies, and the diabody
expression levels in the culture supernatants were investigated by Western blotting. The clone
with the highest expression level was expanded in a nucleic acid-free MEMa medium containing
5 nM MTX, and this was stocked as an overproducing cell line.
30
[14] Large-scale purification of 2D7 diabodies
A subconfluent 2D7DB-producing CHO cell line in a T-125 flask was detached using
Trypsin-EDTA, and then this was transferred to a roller bottle (250 ml of MEMa without
nucleotide + 5% FCS). Four days later, the culture solution was removed, and the cells were
35 washed twice with PBS. The medium was then exchanged to 250 ml of CHO-S-SFMII
medium (GIBCO BRL) to produce a serum-free medium, cells were cultured for three days, and
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then the cell culture supernatant was collected. After removing the dead cells by centrifugation,
this was filtered and used for purification.
Purification of single chain Fv was performed as follows: First, the collected culture
supernatant was applied and adsorbed onto an anti-Flag M2 column. After washing with buffer
5 A (50 mM Tris-HCl pH7.4, 150 mM NaCl, 0.01% Tween 20), single chain Fv was eluted with
buffer B (1 00 mM Glycine pH3.5, 0.01% Tween 20). The collected sample was immediately
neutralized with Tris-HCl pH8.0 so that the final concentration was 25 mM. This was then
used for gel filtration purification by a Superdex 200HR (26/60) column. The dimer fraction of
single chain Fv was collected in PBS containing 0.01% Tween 20. A portion of the collected
10 sample was subjected to SDS electrophoresis and silver staining to confirm that the protein of
interest has been purified, and then this was concentrated to produce a purified authentic sample
of 2D7 diabody.
[15] Cell death induction experiment using 2D7 diabody
15 Various hemocyte cell lines were plated into.24-well plates at 2-5 x 10 5 cells/well.
Purified 2D7DB, or the culture supernatant of COS7 transiently expressing 2D7DB, was added
and cell death was induced. When used, the culture supernatant of COS7 transiently expressing
2D7DB was added so its concentration was 50%. The amount of medium in each well was 0.8
to 1 ml/well. When stimulating Jurkat cells, Con A (WAKO) was added at the time of 2D7DB
20 addition to a final concentration of 2 ug/ml.
Adherent cells (HeLa) were plated into a 6-well plate at 2x 10 5 cells/well, and the cells
were attached by culturing overnight. Subsequently, purified 2D7DB was added to the culture
solution.
Several hours to several days after 2D7DB addition, the suspended cells were collected
25 as they were, and adherent cells were collected after detaching the cells with 1 mM EDTA/PBS.
The cells were then washed with ice-cold PBS, and labeled with Annexin V, which is an
apoptosis marker, and with PI, which is a dead-cell marker, according to the attached instructions
(TACS Annexin V-FITC Apoptosis Detection Kit, TREVIGEN Instructions). The proportion of
stained cells was then measured using flow cytometry (EPICS ELITE, COULTER).
30
[ 1 6] Cell death induction by Actinomycin D
Various hemocyte cell lines were plated into 24-well plates at 2-5 x 10 s cells/well. To
inhibit the initial stage of apoptosis, a caspase inhibitor (Z-VAD-FMK, Promega) was added at a
final concentration of 50 uM, and after incubating for 2.5 hours, cell death was induced. For
35 cell death induction by Actinomycin D, Actinomycin D (Sigma) was added at 1 ug/ml (Jurkat) or
5 ug/ml (ARH77), and for cell death induction by 2D7DB, 2 ug/ml of purified 2D7DB was
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added. Cells were collected 16 hours after cell death induction, and stained using Annexin V
and PI.
[17] Cell growth assay using 2D7 diabody
5 Each type of cells was plated into a 96-well plate at a cell concentration of 1-2 x 10 4
cells/well. 2D7DB was added at an appropriate concentration, and the cell count was
determined after three days of culturing. Viable cell count was determined using WST-8.
More specifically, this reagent was added to the cells at 10 ul/well, and the cells were then
cultured at 37°C for 1 .5 hours. The relative viable cell count was determined by measuring the
10 OD450 using a spectrophotometer. The growth suppression rate was calculated from (1- (OD450
of 2D7DB treated cells / OD450 of 2D7DB untreated cells)) x 1 00.
[18] Detection of DNA fragmentation
ARH77 and Jurkat cells were plated into a 6-well plate so that the cell concentration
15 was 2 x 10 6 cells/well, and cell death was induced by adding purified 2D7DB at a final
concentration of 2 ug/ml, or Actinomycin D at a final concentration of 1 ug/ml (ARH77) or 5
ug/ml (Jurkat) to each well. The control was a well to which nothing was added. After
culturing for 24 hours, the cells were collected, washed once with PBS, and then lysed in a lysis
buffer (10 mM Tris pH7.5, 10 mM EDTA, 0.5% Triton X-100). This was followed by
20 centrifugation to remove the insoluble proteins, and then the material was treated with RNase A
and Proteinase K. A portion of this was then subjected to agarose gel electrophoresis to detect
chromatin DNA fragmentation.
[19] Inhibition of cell death induction by cytochalasin D
25 ARH77 cells were plated into a 24-well plate to achieve a cell concentration of 5 x 10 5
cells/well, and cytochalasin D (Sigma) was added to a final concentration of 20 ug/ml. The
control was a well to which ethanol alone was added. After culturing for one hour, purified
2D7DB was added at various concentrations (0, 200, 500, 1000 ng/ml), and culturing was
continued for another four hours. Cells were then collected, and the proportion of dead cells
30 was detected by staining with PI.
[20] Immunostaining of 2D7DB-treated cells using anti-actin antibody
2D7DB was added at a concentration of 1 ug/ml to cytochalasin D-treated/-untreated
ARH77 cells, and after culturing at 37°C for 15 minutes, the cells were adhered to a slide glass
35 with Cytospin. After immobilizing the cells by immersion in methanol for 1 5 minutes at -20°C,
blocking was performed using a blocking buffer (3% BSA/PBS) at 4°C for one hour. This was
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then reacted with CY3-labeled anti-actin antibody (Sigma) diluted 100-fold in 1% BSA/PBS for
one hour at room temperature, and then the cell nuclei were stained with Hoechst 33258. After
washing several times with PBS, the cells were observed under a confocal laser scanning
microscope (Olympus).
5
[Example 1] Expression analysis of 2D7 antigen in each type of cell line
To determine the cell line that should become the source to produce a cDNA expression
library and the cell line that should become the host, 2D7 antigen expression in each type of
animal cell was analyzed using FACS (Fig. 2A and Fig. 2B). As a result, among
1 0 human-derived hemocyte cells, extremely strong expression of the 2D7 antigen was observed in
lymphocytic tumor cell lines, RPMI8226, U266, and in Jurkat, but expression was found to be
weak in K562. In Ba/F3, FDC-P1 , and HCI-16, which are hemocytes derived from mice,
expression was very weak, perhaps due to differences between the species. Of the adherent
cells, expression was observed in COS7, 293T, and HeLa. Expression was hardly observed in
15 mouse NIH3T3 cells.
From the expression patterns mentioned above, RPMI8226 cells were judged to be
appropriate as a source of a cDNA library to be used for expression cloning, and NIH3T3 cells
were determined to be appropriate as host cells to be used for screening, to which the expression
library is transferred.
20
[Example 2] Cloning of 2D7 antigen
[1] Cloning from a protein
Cell lysates were prepared from RPMI8226 cells and U266 cells, which express the 2D7
antigen, and NIH3T3 cells, which do not express the 2D7 antigen, and immunoprecipitation was
25 performed using the 2D7 antibody. As a result, a molecule (approximately 1 2 kD) that
precipitates specifically in RPMI8226 and U266 cells was observed (Fig. 3). This molecule
was not detected by Western blotting using the 2D7 antibody, but since it is at least reproducibly
precipitated by the 2D7 antibody, it was strongly predicted to be the 2D7 antigen itself, or a
molecule that co-precipitates with the 2D7 antigen.
30 Coomassie staining was performed on this band; it was then cut out and the peptides
were sequenced. As a result, this 12 kD molecule was identified as P2 microglobulin (P2M).
Since p2M is one of the class I MHC protein complexes that associate with HLA class I through
non-covalent bonds, the 2D7 antibody is considered to have co-precipitated it as an HLA
complex. HLA class I comprises the cd and a2 domains required for antigen presentation, and
35 the a3 domain which binds to p2M. Since the 2D7 antibody can co-precipitate the P2M
molecule, it is anticipated that the 2D7 antibody will recognize the al-a2 domains of HLA class
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I as an epitope.
[2] Expression cloning of genes
cDNAs were synthesized using random hexamers from mRNAs purified from the 2D7
5 antigen-expressing cells, RPMI8226. These were inserted into a retrovirus vector, pMX2, and a
retrovirus expression library was constructed. The library titer was investigated, and found to
include a total of 6 x 10 6 clones. Furthermore, the average cDNA length was found to be
approximately 1.5 kb, arrived at by randomly selecting 24 clones from this library and
investigating their insert size using colony PCR. Thus, the produced expression library was
10 judged to be sufficient for use in expression cloning.
Fig. 4A and Fig. 4B show a flow diagram of the screening described below. In the first
screening, 4000 independent clones were used in one pool, and 24 pools (corresponding to
96000 clones) were produced. The plasmids were packaged into retroviruses by transfecting
each plasmid into BOSC23 cells. The resulting viruses derived from each pool were infected
15 into NIH3T3 cells. Three days after infection, the cells were detached, and after staining with
2D7 antibody, expression analysis was performed using FACS. As a result, compared to
NIH3T3 cells infected with viruses derived from an empty vector (control), 2D7-positive cells
were found in 3 of the 24 pools (pools 4, 13, and 21).
Next, pools 4 and 13, which showed positive results in the first screening, were divided
20 into four pools each comprising 1000 independent clones, and a second screening was performed.
As a result, a single clearly positive pool was found from each pool (Fig. 5A, pool 4-4, and pool
13-1). Pool 13-1 was further divided into 21 pools, each comprising 160 independent clones, to
perform a third screening. Two positive pools (Fig. 5B, 13-1-1 1 and 13-1-21) were identified.
Subsequently, pool 13-1-11 was divided into eight pools, each comprising 20 clones, to perform
25 a fourth screening, and a positive pool (Fig. 5C, 13-1-11-5) was obtained.
This pool was spread onto an LB plate, 64 colonies were picked one by one, and each of
these were inoculated to one well of a 96-well plate. The eight clones in the vertical rows were
taken as one pool to produce eight pools (1 to 8), and the eight clones in the horizontal rows
were taken as one pool to produce eight pools (A to H), and a fifth screening was performed.
30 As a result, pools 3, 4, 6, and 8, and pools E, F, and G were positive, thus narrowing down the
positive candidate clones to twelve clones (Fig. 6A). FACS was performed on these twelve
clones, and ultimately four positive clones (3F, 4G, 6E, and 8G) were identified as a single clone
recognized by the 2D7 antibody (Fig. 6B).
As a result of reading the sequence of the insert portion of these clones, all four clones
35 were found to be the full-length cDNA sequence of Human MHC class I HLA-A-6802.
HLA-A is classified into several dozen types of haplotypes. As a result of this cloning,
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the A*6802 haplotype of HLA class I was identified as a 2D7 antigen, but since the 2D7
antibody recognizes a wide variety of cells, the haplotype of HLA class I in the RPMI8226 cells
that were used as the gene source just happened to be A*6802, and the 2D7 antibody was
considered to be an antibody that recognizes any haplotype of HLA class I molecules.
5
[Example 3] Examination of growth inhibitory effect
Several types of leukemia cell lines (K562, Jurkat, and RPMI8226) were used to
investigate whether the 2D7 antibody has a cytocidal effect. The expression level of the 2D7
antigen in the three cell lines is: K562, weakly positive; Jurkat and RPMI8226, strongly positive.
10 K562 and Jurkat cells were plated in the presence or absence of PHA and PMA, and 10
Hg/ml of the 2D7 antibody was added thereto. On observing the cells 24 hours later, weakly
2D7-positive K562 cells did not show obvious differences in their morphology due to the
presence or absence of the 2D7 antibody, however, addition of 2D7 antibody resulted in
significant cell aggregation in Jurkat cells strongly expressing 2D7 (Fig. 7B and Fig. 7C).
15 However, growth inhibition due to addition of the 2D7 antibody was not observed (Fig. 7A).
Growth inhibition due to 2D7 in Jurkat cells activated by PHA and PMA stimulation was also
not observed.
Unexpectedly, addition of 2D7 antibody did not have an obvious effect on the
morphology and growth of the strongly 2D7-positive RPMI8226 cells (Fig. 7D).
20 Next, it was examined whether cytocidal effects can be observed by adding anti-mouse
IgG(Fc) antibody to 2D7 antibody, to cross-link the antibodies. Anti-mouse IgG was added to
Jurkat cells, in the presence or absence of 2D7 antibody. The cells were cultured, and 48 hours
later the cell nuclei were stained with Hoechst33258. Cells were observed for fragmentation of
cell nuclei, which is characteristic of dead cells (Fig. 8). As a result, nuclear fragmentation was
25 observed in Jurkat cells by further cross-linking 2D7 with an antibody, indicating that cell death
was induced.
[Example 4] Cloning of cDNA encoding the 2D7 antibody variable region, and the predicted
diabody structure
30 Primers for the constant regions of the heavy chain and light chain of mouse IgG2b
were produced, and DNA encoding the 2D7 variable region was cloned by 5'RACE method.
The nucleotide sequences of the obtained PCR products are shown in SEQ ID NO: 1 and 3.
A single chain was then constructed based on these sequences. As shown in Fig. 9 and
Fig. 10A, the 2D7 single chain is composed of the leader sequence of the heavy chain, the
35 variable region of the heavy chain, and then across from a 5mer linker (GGGGS), the variable
region of a light chain, followed by a cDNA (SEQ ID NO: 5) encoding a Flag-tag.
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Dimerization of this single chain may cause the 2D7 diabody to form the structure shown in Fig.
10B.
[Example 5] Analysis of the cytotoxic activity of the 2D7 diabody
5 (i) Cytotoxic activity of the 2D7 diabody transiently expressed in COS7
A 2D7 diabody expression vector was transfected into COS7 cells, and the culture
supernatant was collected three days later. The culture supernatant and cell lysate were
subjected to SDS-PAGE, and after performing Western blotting with an anti-Flag-tag antibody, a
2D7 single chain was found to be secreted in the culture supernatant (Fig. 10C).
10 This culture supernatant was added to Jurkat cells at a ratio of 50%. The percentage of
dead cells was measured by staining the cells with PI and Annexin V a few days later. No
significant change in the apoptosis marker was observed in Jurkat cells to which just the
anti-BST-1 antibody and 2D7 antibody (5 |xg/ml each) were added. Furthermore, no particular
change could be observed when using the culture supernatant of COS7 transfected with the
15 vector alone. On the other hand, cell death was clearly induced in Jurkat cells to which the
culture supernatant of COS7 expressing 2D7DB was added (Fig. 1 1 A and Fig. 1 IB).
Next, to investigate the HLA class I A-specific action of this 2D7DB, a similar
experiment was performed using K562 cells, which are known to not express HLA class I A.
As a result, 2D7DB had absolutely no influence on K562 cells, although it showed cell death
20 inducing activity against Jurkat cells (Fig. 12A and Fig. 12B). This strongly supports the idea
that the cell death inducing activity of 2D7DB is an action targeting HLA class I A, which is its
epitope. Furthermore, according to each data, the sensitivity of Jurkat cells towards 2D7DB
was found to be slightly higher in cells stimulated with con A.
Next, the action of 2D7DB on other myeloma cell lines was analyzed. RPMI8226,
25 IL-KM3, U266, and ARH77 were incubated with culture supernatant in which the vector alone
was transfected (control), or with the 2D7DB-expressing COS7 culture supernatant. Two days
later these cultures were double stained with Annexin V and PI, and analyzed using a flow
cytometer. As a result, incubation with 2D7DB was found to significantly induce cell death in
all of the cells (Fig. 13Ato Fig. 13D).
30
(ii) Cytotoxic activity of purified 2D7DB
The growth inhibitory effect of purified 2D7DB on each type of cell line (RPMI8226,
ARH77, U266, and Jurkat) was analyzed. 2D7DB was added at 0, 0.5, 1 .0, and 2.0 ug/ml, and
the number of cells was counted three days later. As a result, 2D7DB was found to inhibit cell
35 growth of these cells in a concentration-dependent manner (Fig. 14).
Purified 2D7DB was then added, and 48 hours later, cells were stained with cell death
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markers, PI and Annexin V, and then analyzed. As in the results obtained when using 2D7DB
transiently expressed in COS7, cell death was induced in Jurkat and ARH77 in a
concentration-dependent manner, and K562 was not affected at all (Fig. 1 5A to Fig. 1 5C).
Furthermore, 48 hours after the addition of 2D7DB to U266 and IL-KM3, significant cell death
5 inducing activity was confirmed (Fig. 16A and Fig. 16B).
On the other hand, although the 2D7 antibody stained the adherent HeLa cells very well,
2D7DB had absolutely no influence under the same conditions (Fig. 15 D). This suggested that
2D7DB may act specifically on suspended cells, such as hemocyte cells.
Next, the time taken for 2D7DB to induce cell death was analyzed. 2 fig/ml of 2D7DB
10 was added to ARH77 and Jurkat cells, cells were collected 12, 24, and 38 hours later, and stained
with a cell death marker. The results showed that cell death was already induced in all cells
twelve hours later (Fig. 17A and Fig. 17B). Therefore, cell death induction was investigated at
earlier times (three and six hours). Surprisingly, it was shown that 2D7DB induces cell death at
least within three hours after its addition (Fig. 1 8A and Fig. 1 8B). These results strongly
15 support the idea that 2D7DB has a very strong cell death-inducing activity. Since 2D7DB
strongly induces cell death, sufficient drug efficacy can be expected even with a short half life in
the blood. Furthermore, safety becomes a concern if the whole antibody has strong cell
death-inducing activity, considering the length of the half life in the blood; however, producing a
diabody can overcome such problems.
20 Next, analyses were performed to determine whether cell death due to 2D7DB is
induced through caspase activation, that is, whether it is apoptosis. As shown in Fig. 19 and
Fig. 20, significant apoptosis was induced when ARH77 and Jurkat cells were treated with the
apoptosis-inducing agent Actinomycin D, and then stained 16 hours later with Annexin V and PI.
After pre-treating cells under these conditions with caspase inhibitor Z-VAD-FMK for 2.5 hours,
25 apoptosis due to Actinomycin D was suppressed. However, cell death induced by 2D7DB was
not inhibited at all by pretreatment with Z-VAD-FMK. These results show that 2D7DB induces
cell death by a mechanism different from the ordinary caspase-mediated apoptosis mechanism.
To confirm this, fragmentation of chromatin DNA, known to be the most characteristic
biochemical change accompanying apoptosis, was also analyzed.
30 ARH77 and Jurkat cells were treated with 2D7DB (2 ug/ml) or Actinomycin D, and
DNAs were collected from the cells 24 hours later and subjected to electrophoresis (Fig. 21).
As a result, DNA fragmentation characteristic of apoptosis had been induced in all cells treated
with Actinomycin D, which is an apoptosis-inducing agent. On the other hand, DNA
fragmentation was not observed at all in 2D7DB-treated cells, even though the concentration of
35 added 2D7DB was absolutely sufficient to induce cell death. These results also strongly
support the idea that cell death due to 2D7DB is an unknown type of cell death, unaccompanied
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by the characteristics of apoptosis.
From the above-mentioned results, cell death due to 2D7DB was found to be caused by
a pathway different from previously known cell death induction mechanisms. Therefore,
further analysis was performed to elucidate the mechanism of cell death induction by 2D7DB.
5 From the experiments described above, when 2D7DB was reacted with the cells, the cell
membranes were often observed to be destroyed under the microscope. Therefore, 2D7DB was
presumed to have some sort of influence on the actin skeleton. In order to examine such a
possibility, an actin polymerization inhibitor (cytochalasin D) was made to act on the cells, and
the influence of 2D7DB on cell death induction activity was analyzed.
10 Cytochalasin D (20 ug/ml) or ethanol alone (control) was added to ARH77 cells, and 1
hour later, 2D7DB was added at various concentrations. After a 4-hour incubation from the
2D7DB addition, cells were collected, PI staining was performed and the percentage of dead
cells was measured (Fig. 22). As a result, pretreatment of cells with cytochalasin D was found
to cause loss of sensitivity towards 2D7DB. These results suggested that 2D7DB causes some
15 kind of effect on the cytoskeletal system, such as actin, to induce cell death by binding to
HLA -class IA, which is the target molecule.
Therefore, cells treated with 2D7DB were stained by the actin antibody, and the
dynamic change of the cytoskeletal system due to 2D7DB addition was analyzed visually.
ARH77 cells were treated with 2D7DB, and 15 minutes later, the cells were immobilized with
20 methanol, and the state of actin (red) in the cells was investigated by immunostaining (Fig. 23).
As a result, compared to the image from those not treated with 2D7DB, significant destruction of
the actin skeleton in the cell due to 2D7DB was observed.
The above-mentioned results strongly suggested that cell death due to 2D7DB may be
caused by destruction of the actin skeleton in cells by 2D7DB bound to HLA class IA. This is a
25 completely new type of cell death induction mechanism that has not been reported to date.
[Example 6] Drug efficacy test for 2D7 diabody using human myeloma animal model
(1) Production of mouse model for human myeloma
A mouse model for human myeloma was produced as follows. ARH 77 cells were
30 prepared to reach 2.5x 1 0 7 cells/ml in RPMI 1 640 medium (GIBCO BRL) supplemented with
10% fetal calf serum (GIBCO BRL), and then 200 |aL of the above-mentioned ARH77 cell
suspension (5x 10 6 cells/mouse) was injected to SCID mice (male, 6 weeks old, Clea Japan)
pretreated the day before with intraperitoneal administration of 0.2 mg of anti-asialo GM1
antibody (Wako Pure Chemicals) from the tail vein.
35
(2) Preparation of the antibody to be administered
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On the day of administration, a 2D7 diabody was prepared at 0.8 mg/ml using
filter-sterilized PBS(-), and this was used as the administration sample.
(3) Antibody administration
5 To the mouse model of human myeloma produced in (1), the administration sample
prepared in (2) was administered through the tail vein at 1 0 ml/kg twice a day for 3 days from
the first day after engraftment of ARH77 cells. As a negative control (vehicle), filter-sterilized
PBS(-) was administered similarly at 10 ml/kg through the tail vein twice a day for 3 days. The
antibody-administered group had 7 animals per group, and the vehicle-administered group had 8
1 0 animals per group.
(4) Human IgG assay of mouse serum
The quantity of human IgG produced by human myeloma cells in the mouse serum was
determined by ELISA described below. 100 uL of goat anti -human IgG antibody
1 5 (BIOSOURCE) diluted to 1 ug/ml with 0. 1 % bicarbonate buffer (pH9.6) was placed into a
96-well plate (Nunc), this was incubated at 4°C overnight, and the antibody was immobilized.
After blocking, mouse serum diluted in a stepwise manner, or as the authentic sample, 100 uL of
human IgG (Cappel) was added, and this was incubated at room temperature for 1 hour. After
washing, 100 uL of a 5000-fold diluted alkaline phosphatase-labeled anti-human IgG antibody
20 (BIOSOURCE) was added, and this was incubated at room temperature for 1 hour. After
washing, substrate solution was added, and after incubation, absorbance at 405 nm was measured
using MICROPLATE READER Model 3550 (BioRad), and the concentration of human IgG in
mouse serum was calculated from the calibration curve obtained from the absorbance of the
authentic human IgG sample.
25
(5) Evaluation of anti-tumor effect
The anti-tumor effect of the 2D7 diabody on a human myeloma mouse model was
evaluated using the change in the amount of human IgG (M protein) produced by the myeloma
cells in mouse serum, and by the survival time. Regarding the change in human IgG level in
30 mouse serum, serum was collected on the 24th day after transplanting the ARH77 cells, and the
human IgG level was measured by the ELISA described above in (4). As a result, the level of
human IgG (M protein) in the serum had increased in the vehicle-administered group to
approximately 74 ug/ml. In contrast, the level in the 2D7 diabody-administered group was
significantly lower than in the control group (P<0.005, unpaired t-test), and 2D7 diabody was
35 shown to very strongly suppress the growth of ARH77 cells (Fig. 24). With regards to survival
time, as shown in Fig. 25, the 2D7 diabody-administered group showed a significant increase in
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survival time compared to the vehicle-administered group.
Accordingly, the 2D7 diabody was shown to have an antitumor effect on the mouse
model of human myeloma. The antitumor effect of the 2D7 diabodies of this invention may be
based on the cell death-inducing action of this antibody.
5
[Example 7] Analysis of the action of 2D7DB on PBMC
The action of 2D7DB on human peripheral blood mononuclear cells (PBMCs) was
analyzed. PBMCs were purified from the peripheral blood of a healthy adult volunteer by
density gradient centrifugation. The PBMCs were plated at 5x 10 5 cells/1 ml/well onto a
10 24-well plate, in the presence or absence of a mitogen. Phytohemagglutinin M (PHA-M, Roche
Diagnostics, final concentration: 10 ug/ml), concanavalin A (ConA, Wako, final concentration:
10 ug/ml), and SAC (Pansorbin Cells, Calbiochem, final concentration: 0.01%) were used as
mitogens. Cells were cultured in a 5% CO2 incubator at 37°C for three days. 24 or 3 hours
before culture was complete, 2D7DB was added to yield a final concentration of 2 ug/ml. After
15 culture was complete, the cells were double stained with Annexin V and PI (Annexin V-FITC
Apoptosis Detection Kit I, Pharmingen), and then analyzed using a flow cytometer (EPICS XL,
Coulter). As a positive control, ARH77 at 2.5x 10 5 cells/1 ml/well was cultured for 24 hours in
the absence of a mitogen, and was reacted with 2D7DB, as for PBMC.
In the case of PBMC, the percentages of dead cells that were both Annexin V and
20 Pi-positive were 29%, 23%, and 25% in the absence of mitogens (in order: no addition, 3-hour
addition, and 24-hour addition of 2D7DB; continued below); 20%, 45%, and 42% in the
presence of PHA-M; 22%, 30%, and 34% in the presence of ConA; and 31%, 38%, and 40% in
the presence of SAC (Figs. 26A to 26D). In the case of ARH77, the percentages were 1 6%,
56%, and 58% (Fig. 26E). These results showed that 2D7DB has hardly any effect on
25 unstimulated PBMC, but induces cell death in a short time with mitogen-activated PBMC.
Industrial Applicability
This invention provides minibodies with high specific activities. By using these
minibodies, adequate drug efficacy can be expected even with a short half life. The minibodies
30 of the present invention are further expected to be able to separate drug efficacy from toxicity.
In addition, since overall cost is reduced, including reducing clinical dose and production cost,
economical problems of concern in the development of antibody pharmaceuticals are also
expected to improve.
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CLAIMS
I. A minibody that recognizes a human leukocyte antigen (HLA).
5 2. The minibody of claim 1 , wherein the HLA is an HLA class I.
3. The minibody of claim 2, wherein the HLA class I is an HLA-A.
4. A minibody derived from a 2D7 antibody.
10
5. The minibody of any one of claims 1 to 4, wherein the minibody is a diabody.
6. A minibody of any one of (a) to (d):
(a) a minibody comprising the amino acid sequence of SEQ ID NO: 6;
(b) a minibody functionally equivalent to the minibody of (a), and comprising an amino
acid sequence with a substitution, insertion, deletion and/or addition of one or more amino acids
in the amino acid sequence of SEQ ID NO: 6;
(c) a minibody comprising the amino acid sequences of CDRs of SEQ ID NOs: 2 and 4;
and
(d) a minibody functionally equivalent to the minibody of (c), and comprising an amino
acid sequence with a substitution, insertion, deletion and/or addition of one or more amino acids
in the amino acid sequence of the CDRs of SEQ ID NOs: 2 and 4.
7. A method for producing an HLA-recognizing antibody having increased activity by
25 converting the HLA-recognizing antibody to a low-molecular-weight antibody.
8. The method of claim 7, wherein the HLA is an HLA class I.
9. The method of claim 8, wherein the HLA class I is an HLA-A.
30
10. A method for producing a 2D7 antibody having increased activity by converting the 2D7
antibody to a low-molecular-weight antibody.
I I . The method of any one of claims 7 to 10, wherein the conversion step comprises
35 conversion to a diabody.
15
20
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12. The method of any one of claims 7 to 11, wherein the activity is a cell death-inducing
activity or a cell growth-suppressing activity.
13. A cell death-inducing agent, comprising as an active ingredient the minibody of any one of
5 claims 1 to 6, the minibody produced by the method of any one of claims 7 to 12, or a 2D7
antibody.
14. The cell death-inducing agent of claim 13 that induces cell death of a B cell or T cell.
10 15. The cell death-inducing agent of claim 14, wherein the B cell or T cell is an activated B cell
or activated T cell.
1 6. A cell growth-suppressing agent comprising as an active ingredient the minibody of any
one of claims 1 to 6, the minibody produced by the method of any one of claims 7 to 12, or a
15 2D7 antibody.
17. An antitumor agent comprising as an active ingredient the minibody of any one of claims
1 to 6, the minibody produced by the method of any one of claims 7 to 12, or a 2D7 antibody.
20 1 8. The antitumor agent of claim 1 7, wherein the tumor is a blood tumor.
19. A therapeutic agent for an autoimmune disease, wherein the therapeutic agent comprises as
an active ingredient the minibody of any one of claims 1 to 6, the minibody produced by the
method of any one of claims 7 to 12, or a 2D7 antibody.
25
20. The cell death-inducing agent of any one of claims 13 to 15, wherein the antibody is a
diabody.
21. The cell growth-suppressing agent of claim 16, wherein the antibody is a diabody.
30
22. The antitumor agent of claim 17 or 18, wherein the antibody is a diabody.
23. The therapeutic agent for autoimmune disease of claim 19, wherein the antibody is a
diabody.
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ABSTRACT
To identify antigens of the 2D7 antibody, the present inventors cloned the 2D7 antigen.
The results suggested that the 2D7 antigen is an HLA class I molecule. Based on this finding,
5 the present inventors examined whether the 2D7 antibody has cell death-inducing activity.
Nuclei fragmentation was observed when the 2D7 antibody was cross-linked with another
antibody, indicating that cell-death was induced. Further, diabodies of the 2D7 antibody were
found to have very strong cell death-inducing activities, even without the addition of another
antibody. These results indicate that minibodies of an HLA-recognizing antibody can be used
10 as cell death-inducing agents.
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